Polymorphic markers of prostate carcinoma tumor antigen-1(PCTA-1)

ABSTRACT

The invention concerns the geNo.mic sequence and cDNA sequences of the PCTA-1 gene. The invention also concerns biallelic markers of the PCTA-1 gene and the association established between these markers and prostate cancer. The invention provides means to determine the predisposition of individuals to prostate cancer as well as means for the diagNo.sis of prostate cancer and for the progNo.sis/detection of an eventual treatment response to agents acting against prostate cancer.

RELATED APPLICATIONS

The present application claims priority from U.S. Provisional Patent Application Serial No. 60/088,187, filed Jun. 5, 1998, and U.S. Provisional Patent Application Serial No. 60/102,324, filed Sep. 28, 1998, the disclosures of which are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The invention concerns the genomic and cDNA sequences of the PCTA-1 gene, biallelic markers of the PCTA-1 gene and the association established between these markers and prostate cancer. The invention provides means to determine the predisposition of individuals to prostate cancer as well as means for the diagnosis of this cancer and for the prognosis/detection of an eventual treatment response to therapeutic agents acting against prostate cancer.

BACKGROUND OF THE INVENTION Prostate Cancer

The incidence of prostate cancer has dramatically increased over the last decades. It averages 30-50/100,000 males in Western European countries as well as within the US White male population. In these countries, it has recently become the most commonly diagnosed malignancy, being one of every four cancers diagnosed in American males. Prostate cancer's incidence is very much population specific, since it varies from 2/100,000 in China, to over 80/100,000 among African-American males.

In France, the incidence of prostate cancer is 35/100,000 males and it is increasing by 10/100,000 per decade. Mortality due to prostate cancer is also growing accordingly. It is the second cause of cancer death among French males, and the first one among French males aged over 70. This makes prostate cancer a serious burden in terms of public health.

Prostate cancer is a latent disease. Many men carry prostate cancer cells without overt signs of disease. Autopsies of individuals dying of other causes show prostate cancer cells in 30% of men at age 50 and in 60% of men at age 80. Furthermore, prostate cancer can take up to 10 years to kill a patient after the initial diagnosis.

The progression of the disease usually goes from a well-defined mass within the prostate to a breakdown and invasion of the lateral margins of the prostate, followed by metastasis to regional lymph nodes, and metastasis to the bone marrow. Cancer metastasis to bone is common and often associated with uncontrollable pain.

Unfortunately, in 80% of cases, diagnosis of prostate cancer is established when the disease has already metastasized to the bones. Of special interest is the observation that prostate cancers frequently grow more rapidly in sites of metastasis than within the prostate itself.

Early-stage diagnosis of prostate cancer mainly relies today on Prostate Specific Antigen (PSA) dosage, and allows the detection of prostate cancer seven years before clinical symptoms become apparent. The effectiveness of PSA dosage diagnosis is however limited, due to its inability to discriminate between malignant and non-malignant affections of the organ and because not all prostate cancers give rise to an elevated serum PSA concentration. Furthermore, PSA dosage and other currently available approaches such as physical examination, tissue biopsy and bone scans are of limited value in predicting disease progression.

Therefore, there is a strong need for a reliable diagnostic procedure which would enable a more systematic early-stage prostate cancer prognosis.

Although an early-stage prostate cancer prognosis is important, the possibility of measuring the period of time during which treatment can be deferred is also interesting as currently available medicaments are expensive and generate important adverse effects. However, the aggressiveness of prostate tumors varies widely. Some tumors are relatively aggressive, doubling every six months whereas others are slow-growing, doubling once every five years. In fact, the majority of prostate cancers grow relatively slowly and never becomes clinically manifest. Very often, affected patients are among the elderly and die from another disease before prostate cancer actually develops. Thus, a significant question in treating prostate carcinoma is how to discriminate between tumors that will progress and those that will not progress during the expected lifetime of the patient.

Hence, there is also a strong need for detection means which may be used to evaluate the aggressiveness or the development potential of prostate cancer tumors once diagnosed.

Furthermore, at the present time, there is no means to predict prostate cancer susceptibility. It would also be very beneficial to detect individual susceptibility to prostate cancer. This could allow preventive treatment and a careful follow up of the development of the tumor.

A further consequence of the slow growth rate of prostate cancer is that few cancer cells are actively dividing at any one time, rendering prostate cancer generally resistant to radiation and chemotherapy. Surgery is the mainstay of treatment but it is largely ineffective and removes the ejaculatory ducts, resulting in impotence. Oral oestrogens and luteinizing releasing hormone analogs are also used for treatment of prostate cancer. These hormonal treatments provide marked improvement for many patients, but they only provide temporary relief. Indeed, most of these cancers soon relapse with the development of hormone-resistant tumor cells and the oestrogen treatment can lead to serious cardiovascular complications. Consequently, there is a strong need for preventive and curative treatment of prostate cancer.

Efficacy/tolerance prognosis could be precious in prostate cancer therapy. Indeed, hormonal therapy, the main treatment currently available, presents important side effects. The use of chemotherapy is limited because of the small number of patients with chemosensitive tumors. Furthermore the age profile of the prostate cancer patient and intolerance to chemotherapy make the systematic use of this treatment very difficult.

Therefore, a valuable assessment of the eventual efficacy of a medicament to be administered to a prostate cancer patient as well as the patient's eventual tolerance to it may allow the benefit/risk ratio of prostate cancer treatment to be enhanced.

Prostate Carcinoma Tumor Antigen-1 (PCTA-1)

WO 96/21671 describes a new protein, named PCTA-1. The document describes the cloning and sequencing of a cDNA encoding PCTA-1 (GenBank L78132). This cDNA has 3.85 kb in length and presents about 80% sequence homology with rat galectin-8.

WO 96/21671 mentions that the PCTA-1 protein retains a number of conserved structural motifs that are found in most members of the galectin gene family. On the basis of its predicted amino acid sequence, PCTA-1 is said to appear to be a human homologue of rat galectin-8. The galectins display wide tissue distribution, clear developmental regulation, and differential levels in specific tissues, supporting the hypothesis that they contribute to many physiologically important processes in mammalian cells. Of direct relevance to cancer is the finding that the galectins can mediate both cell-cell and cell-matrix interactions.

SUMMARY OF THE INVENTION

The inventors have characterized the genomic sequence of the PCTA-1 gene, including its regulatory regions, and, through an association study, have shown that alleles of some biallelic markers of PCTA-1 are associated with prostate cancer.

Therefore, the present invention concerns the identification and characterization of the genomic sequence of the PCTA-1 gene, of new cDNA sequences and the proteins encoded by these cDNAs. The invention also concerns biallelic markers located in such sequences, as well as the selection of significant polymorphisms associated with prostate cancer.

Oligonucleotide probes and primers hybridizing specifically with a genomic sequence of PCTA-1 are also part of the invention. A further object of the invention consists of recombinant vectors comprising any of the nucleic acid sequences described in the present invention, and in particular of recombinant vectors comprising the regulatory region of PCTA-1 or a sequence encoding a PCTA-1 protein, as well as cell hosts comprising said nucleic acid sequences or recombinant vectors.

The selected polymorphisms are used in the design of assays for the reliable detection of genetic susceptibility to prostate cancer, of an early onset of prostate cancer, of the aggressiveness of prostate cancer tumors, of a modified or forthcoming expression of the PCTA-1 gene, of a modified or forthcoming production of the PCTA-1 protein, or of the production of a modified PCTA-1 protein. They can be used for diagnosis, staging, prognosis, and monitoring of such a disease, which processes can be further included within treatment approaches. The selected polymorphisms can also be used in the design of drug screening protocols to provide an accurate and efficient evaluation of the therapeutic and side-effect potential of new or already existing medicaments.

The invention also encompasses methods of screening of molecules which modulate or inhibit the expression of the PCTA-1 gene and more preferably of agent acting against prostate cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a diagram of the PCTA-1 gene with an indication of the relative position of the biallelic markers of the present invention. The upper line refers to the genomic sequence of PCTA-1. The middle line refers to the alternative cDNA comprising the exon 6bis with the biallelic markers localization. The lower line refers the PCTA-1 protein with the polymorphic amino acids due to the biallelic markers. {circle around (⋆)} refers to frequent SNP (detected on pool of hundred DNA).

FIG. 1B is a diagram of the 3 alternative cDNAs of PCTA-1.

FIG. 2 is a graph demonstrating the association between some of the biallelic markers of the invention and prostate cancer with the absolute value of the logarithm (base 10) of the p-value of the chi-square values for each marker shown on the y-axis and a rough estimate of the position of each marker with respect to the PCTA-1 gene elements on the x-axis.

FIG. 3 is a block diagram of an exemplary computer system.

FIG. 4 is a flow diagram illustrating one embodiment of a process 200 for comparing a new nucleotide or protein sequence with a database of sequences in order to determine the homology levels between the new sequence and the sequences in the database.

FIG. 5 is a flow diagram illustrating one embodiment of a process 250 in a computer for determining whether two sequences are homologous.

FIG. 6 is a flow diagram illustrating one embodiment of an identifier process 300 for detecting the presence of a feature in a sequence.

FIGS. 7A-D is an alignment of the mouse and human PCTA-1 proteins. The amino acid sequences provided in the alignment are presented as SEQ ID NOs: 13 (leg2), 14 (leg1), 15 (PCTA), 16 (PCTA.var), 17 (PCTA.mus), 18 (gal9-1), 19 (gal), 20 (leg7), 21 (gal4), and (consenus sequence).

BRIEF DESCRIPTION OF THE SEQUENCES PROVIDED IN THE SEQUENCE LISTING

SEQ ID No 1 contains a genomic sequence of PCTA-1 comprising the 5′ regulatory region (upstream untranscribed region), the exons (0, 1, 2, 3, 4, 5, 6, 6bis, 7, 8, 9, 9bis, and 9ter) and introns, and the 3′ regulatory region (downstream untranscribed region).

SEQ ID No 2 contains a cDNA sequence of PCTA-1 comprising the exons 0, 1, 2, 3, 4, 5, 6, 7, 8, and 9.

SEQ ID No 3 contains a cDNA sequence of PCTA-1 comprising the exons 0, 1, 2, 3, 4, 5, 6, 6bis, 7, 8, and 9.

SEQ ID No 4 contains a cDNA sequence of PCTA-1 comprising the exons 0, 1, 2, 3, 4, 5, 6, 7, 8, 9bis and 9ter.

SEQ ID No 5 contains the amino acid sequence encoded by the cDNA of SEQ ID No 2.

SEQ ID No 6 contains the amino acid sequence encoded by the cDNA of SEQ ID No 3.

SEQ ID No 7 contains the amino acid sequence encoded by the cDNA of SEQ ID No 4.

SEQ ID No 8 contains a murine cDNA sequence of PCTA-1.

SEQ ID No 9 contains the amino acid sequence encoded by the cDNA of SEQ ID No 8.

SEQ ID No 10 contains a primer containing the additional PU 5′ sequence described further in Example 2.

SEQ ID No 11 contains a primer containing the additional RP 5′ sequence described further in Example 2.

SEQ ID NO: 12 is a version of the polynucleotide sequence of the PCTA-1 gene of SEQ ID NO: 1 that provides symbols appropriate for indicated allelic substitutions at nucleotide positions 402, 67092, 68525, 82234, and 82393. SEQ ID NO: 12.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Before describing the invention in greater detail, the following definitions are set forth to illustrate and define the meaning and scope of the terms used to describe the invention herein.

The term “PCTA-1 gene” is intended to define an entity which can comprise some or all the following elements: exons, introns, promoter, regulatory regions, 5′UTR, 3′UTR and regions never transcribed and located either upstream or downstream of the coding sequence of PCTA-1 . The term “PCTA-1 gene”, when used herein, encompasses genomic, mRNA and cDNA sequences encoding a PCTA-1 protein.

The term “heterologous protein”, when used herein, is intended to designate any protein or polypeptide other than the PCTA-1 protein. More particularly, the heterologous protein is a compound which can be used as a marker in further experiments with a PCTA-1 regulatory region or as a toxin to certain cells in which it is intended to be produced, preferably a toxin to prostate cancer cells.

As used herein, the term “toxin gene” refers to a polynucleotide sequence which encodes a polypeptide that, when expressed in a eukaryotic cell, typically a mammalian cell, kills or disables the cell or causes the cell to exhibit apoptosis, cytostasis or senescence.

The term “isolated” requires that the material be removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition, and still be isolated in that the vector or composition is not part of its natural environment.

The term “purified” does not require absolute purity; rather, it is intended as a relative definition. Purification of starting material or natural material to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. As an example, purification from 0.1% concentration to 10% concentration is two orders of magnitude. The term “purified” is used herein to describe a polynucleotide or polynucleotide vector of the invention which has been separated from other compounds including, but not limited to other nucleic acids, carbohydrates, lipids and proteins (such as the enzymes used in the synthesis of the polynucleotide), or the separation of covalently closed polynucleotides from linear polynucleotides. A polynucleotide is substantially pure when at least about 50%, preferably 60 to 75% of a sample exhibits a single polynucleotide sequence and conformation (linear versus covalently closed). A substantially pure polynucleotide typically comprises about 50%, preferably 60 to 90% weight/weight of a nucleic acid sample, more usually about 95%, and preferably is over about 99% pure. Polynucleotide purity or homogeneity is indicated by a number of means well known in the art, such as agarose or polyacrylamide gel electrophoresis of a sample, followed by visualizing a single polynucleotide band upon staining the gel. For certain purposes higher resolution can be provided by using HPLC or other means well known in the art.

As used interchangeably herein, the terms “nucleic acids”, “oligonucleotides”, and “Polynucleotides” include RNA, DNA, or RNA/DNA hybrid sequences of more than one nucleotide in either single chain or duplex form. The term “nucleotide” as used herein as an adjective to describe molecules comprising RNA, DNA, or RNA/DNA hybrid sequences of any length in single-stranded or duplex form. The term “nucleotide” is also used herein as a noun to refer to individual nucleotides or varieties of nucleotides, meaning a molecule, or individual unit in a larger nucleic acid molecule, comprising a purine or pyrimidine, a ribose or deoxyribose sugar moiety, and a phosphate group, or phosphodiester linkage in the case of nucleotides within an oligonucleotide or polynucleotide. Although the term “nucleotide” is also used herein to encompass “modified nucleotides” which comprise at least one modifications (a) an alternative linking group, (b) an analogous form of purine, (c) an analogous form of pyrimidine, or (d) an analogous sugar, for examples of analogous linking groups, purine, pyrimidines, and sugars see for example PCT publication No. WO 95/04064. This may be especially oligonucleotides with α or β anomers, oligonucleotides with inter-nucleotide linkage of the phosphorothioate or methyl phosphonate type, or alternatively oligothionucleotide. The polynucleotide sequences of the invention may be prepared by any known method, including synthetic, recombinant, ex vivo generation, or a combination thereof, as well as utilizing any purification methods known in the art.

Throughout the present specification, the expression “nucleotide sequence” may be employed to designate indifferently a polynucleotide or a nucleic acid. More precisely, the expression “nucleotide sequence” encompasses the nucleic material itself and is thus not restricted to the sequence information (i.e. the succession of letters chosen among the four base letters) that biochemically characterizes a specific DNA or RNA molecule.

A “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell required to initiate the specific transcription of a gene.

A sequence which is “overably linked” to a regulatory sequence such as a promoter means that said regulatory element is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the nucleic acid of interest. As used herein, the term “operably linked” refers to a linkage of polynucleotide elements in a functional relationship. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. More precisely, two DNA molecules (such as a polynucleotide containing a promoter region and a polynucleotide encoding a desired polypeptide or polynucleotide) are said to be “operably linked” if the nature of the linkage between the two polynucleotides does not (1) result in the introduction of a frame-shift mutation or (2) interfere with the ability of the polynucleotide containing the promoter to direct the transcription of the coding polynucleotide.

The term “primer” denotes a specific oligonucleotide sequence which is complementary to a target nucleotide sequence and used to hybridize to the target nucleotide sequence. A primer serves as an initiation point for nucleotide polymerization catalyzed by either DNA polymerase, RNA polymerase or reverse transcriptase.

The term “probe” denotes a defined nucleic acid segment (or nucleotide analog segment, e.g., polynucleotide as defined herein) which can be used to identify a specific polynucleotide sequence present in samples, said nucleic acid segment comprising a nucleotide sequence complementary of the specific polynucleotide sequence to be identified.

The terms “base paired” and “Watson & Crick base paired” are used interchangeably herein to refer to nucleotides which can be hydrogen bonded to one another by virtue of their sequence identities in a manner like that found in double-helical DNA with thymine or uracil residues linked to adenine residues by two hydrogen bonds and cytosine and guanine residues linked by three hydrogen bonds (See Stryer, L., Biochemistry, 4^(th) edition, 1995).

The terms “complementary” or “complement thereof” are used herein to refer to the sequences of polynucleotides which are capable of forming Watson & Crick base pairing with another specified polynucleotide throughout the entirety of the complementary region. For the purpose of the present invention, a first polynucleotide is deemed to be complementary to a second polynucleotide when each base in the first polynucleotide is paired with its complementary base. Complementary bases are, generally, A and T (or A and U), or C and G. “Complement” is used herein as a synonym of “complementary polynucleotide”, “complementary nucleic acid” and “complementary nucleotide sequence”. These terms are applied to pairs of polynucleotides based solely upon their sequences and not any particular set of conditions under which the two polynucleotides would actually bind.

The term “Volypeptide” refers to a polymer of amino acids without regard to the length of the polymer, thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not specify or exclude post-expression modifications of polypeptides, for example, polypeptides which include the covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed by the term polypeptide. Also included within the definition are polypeptides which contain one or more analogs of an amino acid (including, for example, non-naturally occurring amino acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.

The term “recombinant polypeptide” is used herein to refer to polypeptides that have been artificially designed and which comprise at least two polypeptide sequences that are not found as contiguous polypeptide sequences in their initial natural environment, or to refer to polypeptides which have been expressed from a recombinant polynucleotide.

The term “purified” is used herein to describe a polypeptide of the invention which has been separated from other compounds including, but not limited to nucleic acids, lipids, carbohydrates and other proteins. A polypeptide is substantially pure when at least about 50%, preferably 60 to 75% of a sample exhibits a single polypeptide sequence. A substantially pure polypeptide typically comprises about 50%, preferably 60 to 90% weight/weight of a protein sample, more usually about 95%, and preferably is over about 99% pure. Polypeptide purity or homogeneity is indicated by a number of means well known in the art, such as agarose or polyacrylamide gel electrophoresis of a sample, followed by visualizing a single polypeptide band upon staining the gel. For certain purposes higher resolution can be provided by using HPLC or other means well known in the art.

As used herein, the term “non-human animal” refers to any non-human vertebrate, birds and more usually mammals, preferably primates, farm animals such as swine, goats, sheep, donkeys, and horses, rabbits or rodents, more preferably rats or mice. As used herein, the term “animal” is used to refer to any vertebrate, preferable a mammal. Both the terms “animal” and “mammal” expressly embrace human subjects unless preceded with the term “non-human”.

As used herein, the term “antibody” refers to a polypeptide or group of polypeptides which are comprised of at least one binding domain, where an antibody binding domain is formed from the folding of variable domains of an antibody molecule to form three-dimensional binding spaces with an internal surface shape and charge distribution complementary to the features of an antigenic determinant of an antigen, which allows an immunological reaction with the antigen. Antibodies include recombinant proteins comprising the binding domains, as wells as fragments, including Fab, Fab′, F(ab)₂, and F(ab′)₂ fragments.

As used herein, an “antigenic determinant” is the portion of an antigen molecule, in this case a PCTA-1 polypeptide, that determines the specificity of the antigen-antibody reaction. An “epitope” refers to an antigenic determinant of a polypeptide. An epitope can comprise as few as 3 amino acids in a spatial conformation which is unique to the epitope. Generally an epitope consists of at least 6 such amino acids, and more usually at least 8-10 such amino acids. Methods for determining the amino acids which make up an epitope include x-ray crystallography, 2-dimensional nuclear magnetic resonance, and epitope mapping e.g. the Pepscan method described by Geysen et al. 1984; PCT Publication No. WO 84/03564; and PCT Publication No. WO 84/03506, the disclosures of which are incorporated herein by reference in their entireties.

The term “allele” is used herein to refer to variants of a nucleotide sequence. A biallelic polymorphism has two forms. Diploid organisms may be homozygous or heterozygous for an allelic form.

The term “heterozyposity rate” is used herein to refer to the incidence of individuals in a population which are heterozygous at a particular allele. In a biallelic system, the heterozygosity rate is on average equal to 2P_(a)(1-P_(a)), where P_(a) is the frequency of the least common allele. In order to be useful in genetic studies, a genetic marker should have an adequate level of heterozygosity to allow a reasonable probability that a randomly selected person will be heterozygous.

The term “genotype” as used herein refers the identity of the alleles present in an individual or a sample. In the context of the present invention, a genotype preferably refers to the description of the biallelic marker alleles present in an individual or a sample. The term “genotyping” a sample or an individual for a biallelic marker consists of determining the specific allele or the specific nucleotide carried by an individual at a biallelic marker.

The term “mutation” as used herein refers to a difference in DNA sequence between or among different genomes or individuals which has a frequency below 1%.

The term “haplotype” refers to a combination of alleles present in an individual or a sample. In the context of the present invention, a haplotype preferably refers to a combination of biallelic marker alleles found in a given individual and which may be associated with a phenotype.

The term “polymorphism” as used herein refers to the occurrence of two or more alternative genomic sequences or alleles between or among different genomes or individuals. “Polymorphic” refers to the condition in which two or more variants of a specific genomic sequence can be found in a population. A “polymorphic site” is the locus at which the variation occurs. A single nucleotide polymorphism is a single base pair change. Typically a single nucleotide polymorphism is the replacement of one nucleotide by another nucleotide at the polymorphic site. Deletion of a single nucleotide or insertion of a single nucleotide, also give rise to single nucleotide polymorphisms. In the context of the present invention “single nucleotide polymorphism” preferably refers to a single nucleotide substitution. However, the polymorphism can also involve an insertion or a deletion of at least one nucleotide, preferably between 1 and 5 nucleotides. The nucleotide modification can also involve the presence of several adjacent single base polymorphisms. This type of nucleotide modification is usually called a “variable motif”. Generally, a “variable motif” involves the presence of 2 to 10 adjacent single base polymorphisms. In some instances, series of two or more single base polymorphisms can be interrupted by single bases which are not polymorphic. This is also globally considered to be a “variable motif”. Typically, between different genomes or between different individuals, the polymorphic site may be occupied by two different nucleotides.

The term “biallelic polymorphism” and “biallelic marker” are used interchangeably herein to refer to a polymorphism, usually a single nucleotide, having two alleles at a fairly high frequency in the population. A “biallelic marker allele” refers to the nucleotide variants present at a biallelic marker site. Typically, the frequency of the less common allele of the biallelic markers of the present invention has been validated to be greater than 1%, preferably the frequency is greater than 10%, more preferably the frequency is at least 20% (i.e. heterozygosity rate of at least 0.32), even more preferably the frequency is at least 30% (i.e. heterozygosity rate of at least 0.42). A biallelic marker wherein the frequency of the less common allele is 30% or more is termed a “high quality biallelic marker”.

As used herein the terminology “defining a biallelic marker” means that a sequence includes a polymorphic base from a biallelic marker. The sequences defining a biallelic marker may be of any length consistent with their intended use, provided that they contain a polymorphic base from a biallelic marker. The sequence has between 2 and 100, preferably between 20, 30, or 40 and 60, and more preferably about 47 nucleotides in length. Likewise, the term “marker” or “biallelic marker” requires that the sequence is of sufficient length to practically (although not necessarily unambiguously) identify the polymorphic allele, which usually implies a length of at least 4, 5, 6, 10, 15, 20, 25, or 40 nucleotides.

As used herein the term “PCTA-1-related biallelic marker” or “biallelic marker of the PCTA-1 gene” relates to a set of biallelic markers in linkage disequilibrium with the PCTA-1 gene. The term PCTA-1-related biallelic marker encompasses all of the biallelic markers A1 to A125 disclosed in Table 2.

The location of nucleotides in a polynucleotide with respect to the center of the polynucleotide are described herein in the following manner. When a polynucleotide has an odd number of nucleotides, the nucleotide at an equal distance from the 3′ and 5′ ends of the polynucleotide is considered to be “at the center” of the polynucleotide, and any nucleotide immediately adjacent to the nucleotide at the center, or the nucleotide at the center itself is considered to be “within 1 nucleotide of the center.” With an odd number of nucleotides in a polynucleotide any of the five nucleotides positions in the middle of the polynucleotide would be considered to be within 2 nucleotides of the center, and so on. When a polynucleotide has an even number of nucleotides, there would be a bond and not a nucleotide at the center of the polynucleotide. Thus, either of the two central nucleotides would be considered to be “within 1 nucleotide of the center” and any of the four nucleotides in the middle of the polynucleotide would be considered to be “within 2 nucleotides of the center”, and so on. For polymorphisms which involve the substitution, insertion or deletion of 1 or more nucleotides, the polymorphism, allele or biallelic marker is “at the center” of a polynucleotide if the difference between the distance from the substituted, inserted, or deleted polynucleotides of the polymorphism and the 3′ end of the polynucleotide, and the distance from the substituted, inserted, or deleted polynucleotides of the polymorphism and the 5′ end of the polynucleotide is zero or one nucleotide. If this difference is 0 to 3, then the polymorphism is considered to be “within 1 nucleotide of the center.” If the difference is 0 to 5, the polymorphism is considered to be “within 2 nucleotides of the center.” If the difference is 0 to 7, the polymorphism is considered to be “within 3 nucleotides of the center,” and so on.

The terms “trait” and “phenotype” are used interchangeably herein and refer to any visible, detectable or otherwise measurable property of an organism such as symptoms of, or susceptibility to a disease for example. Preferably, the term “trait” or “phenotype”, when used herein, encompasses, but is not limited to, prostate cancer, an early onset of prostate cancer, a beneficial response to or side effects related to treatment or a vaccination against prostate cancer, a susceptibility to prostate cancer, the level of aggressiveness of prostate cancer tumors, a modified or forthcoming expression of the PCTA-1 gene, a modified or forthcoming production of the PCTA-1 protein, or the production of a modified PCTA-1 protein. However, the term “trait” or “phenotype” can refer to other types of cancer.

The term “susceptibility to prostate cancer” is used herein to designate a strong likelihood for an individual to develop in his lifetime a form of prostate cancer, particularly a form of prostate cancer in which a PCTA-1 protein is expressed. This likelihood is strongly related to the association established between the biallelic markers of the present invention and prostate cancer or other more specific characteristics which can lead to the development of the prostate cancer such as the modified expression of the PCTA-1 gene, the modified production of the PCTA-1 protein or the production of a modified PCTA-1 protein.

The term “aggressiveness” of prostate cancer tumors refers to the metastatic potential of these tumors.

The term “treatment of prostate cancer” when used herein is intended to designate the administration of substances either for prophylactic or curative purposes. When administered for prophylactic purposes, the treatment is provided in advance of the appearance of biologically or clinically significant cancer symptoms. When administered for curative purposes, the treatment is provided to attenuate the pathological symptoms of prostate cancer, to decrease the size or growth of cancer tumors or metastases or to remove them.

The terms “an agent acting against prostate cancer” refers to any drug or compound that is capable of reducing the growth rate, rate of metastasis, or viability of tumor cells in a mammal, is capable of reducing the size or eliminating tumors in a mammal, or is capable of increasing the average life span of a mammal or human with cancer. Agents acting against prostate cancer also include compounds which are able to reduce the risk of cancer developing in a population, particularly a high risk population. Examples of agents acting against prostate cancer include hormonal therapeutic agents (for example, medroxyprogesterone acetate, estramustine phosphate, gonadotrophin releasing hormone (GnRH) agonists, anti-androgens such as flutamide, nilutamide, groserelin, and cyprosterone acetate, anti-gonadotropic agents such as stilboestrol and other oestrogenic agents, progestogens such as megestrol acetate) or chemotherapeutic agents (for example, carboplatin, cisplatin, methotrexate, mitomycin, epirubicin, vinblastine, 5-fluorouracyl, mitozantrone, cyclophosphamide, interferon, N-(4-hydroxyphenyl) retinamide (4HPR)). These agents can be used in combination.

The term “side effects to an agent acting against prostate cancer” refers to adverse effects of therapy resulting from extensions of the principal pharmacological action of the drug or to idiosyncratic adverse reactions resulting from an interaction of the drug with unique host factors. These side effects include, but are not limited to, adverse reactions such as dermatological, hematological or hepatological toxicities and further includes gastric and intestinal ulceration, disturbance in platelet function, renal injury, nephritis, vasomotor rhinitis with profuse watery secretions, angioneurotic edema, generalized urticaria, and bronchial En asthma to laryngeal edema and bronchoconstriction, hypotension, sexual dysfunction, and shock. More particularly, the side effects can be nausea/vomiting, cardiovascular side effects such as deep vein thrombosis and fluid retention, and gynaecomastia.

The term “response to an agent acting against prostate cancer” refers to drug efficacy, including but not limited to ability to metabolize a compound, to the ability to convert a pro-drug to an active drug, and to the pharmacokinetics (absorption, distribution, elimination) and the pharmacodynamics (receptor-related) of a drug in an individual.

In the context of the present invention, a “positive response” to a medicament can be defined as comprising a reduction of the symptoms related to the disease, an increase of survival time or condition to be treated.

In the context of the present invention, a “negative response” to a medicament can be defined as comprising either a lack of positive response to the medicament which does not lead to a symptom reduction or an increase of survival time, or which leads to a side-effect observed following administration of the medicament.

Variants and Fragments

1-Polynucleotides

The invention also relates to variants and fragments of the polynucleotides described herein, particularly of a PCTA-1 gene containing one or more biallelic markers according to the invention.

Variants of polynucleotides, as the term is used herein, are polynucleotides that differ from a reference polynucleotide. A variant of a polynucleotide may be a naturally occurring variant such as a naturally occurring allelic variant, or it may be a variant that is not known to occur naturally. Such non-naturally occurring variants of the polynucleotide may be made by mutagenesis techniques, including those applied to polynucleotides, cells or organisms. Generally, differences are limited so that the nucleotide sequences of the reference and the variant are closely similar overall and, in many regions, identical.

Variants of polynucleotides according to the invention include, without being limited to, nucleotide sequences which are at least 95% identical to a polynucleotide selected from the group consisting of the nucleotide sequences of SEQ ID Nos 1, 2, 3, 4, 8 or to any polynucleotide fragment of at least 8 consecutive nucleotides of a polynucleotide selected from the group consisting of the nucleotide sequences of SEQ ID Nos 1, 2, 3, 4, 8, and preferably at least 99% identical, more particularly at least 99.5% identical, and most preferably at least 99.8% identical to a polynucleotide selected from the group consisting of the nucleotide sequences of SEQ ID Nos 1, 2, 3, 4, 8 or to any polynucleotide fragment of at least 8 consecutive nucleotides of a polynucleotide selected from the group consisting of the nucleotide sequences of SEQ ID Nos 1, 2, 3, 4, 8.

Nucleotide changes present in a variant polynucleotide may be silent, which means that they do not alter the amino acids encoded by the polynucleotide. However, nucleotide changes may also result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. The substitutions, deletions or additions may involve one or more nucleotides. The variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions.

In the context of the present invention, particularly preferred embodiments are those in which the polynucleotides encode polypeptides which retain substantially the same biological function or activity as the mature PCTA-1 protein, or those in which the polynucleotides encode polypeptides which maintain or increase a particular biological activity, while reducing a second biological activity.

A polynucleotide fragment is a polynucleotide having a sequence that entirely is the same as part but not all of a given nucleotide sequence, preferably the nucleotide sequence of a PCTA-1 gene, and variants thereof. The fragment can be a portion of an exon or of an intron of a PCTA-1 gene. It can also be a portion of the regulatory sequences of the PCTA-1 gene, preferably of the promoter. Preferably, such fragments comprise at least one of the biallelic markers A1 to A125, and the complements thereof, or a biallelic marker in linkage disequilibrium therewith.

Such fragments may be “free-standing”, i.e. not part of or fused to other polynucleotides, or they may be comprised within a single larger polynucleotide of which they form a part or region. However, several fragments may be comprised within a single larger polynucleotide.

As representative examples of polynucleotide fragments of the invention, there may be mentioned those which have from about 4, 6, 8, 15, 20, 25, 40, 10 to 30, 30 to 55, 50 to 100, 75 to 100 or 100 to 200 nucleotides in length. Preferred are those fragments having about 47 nucleotides in length, such as those of P1 to P125 and the complementary sequences thereto, and containing at least one of the biallelic markers of the PCTA-1 gene which are described herein. It will of course be understood that the polynucleotides P1 to P125 and the complementary sequences thereto can be shorter or longer, although it is preferred that they at least contain the biallelic marker of the primer which can be located at one end of the fragment.

2-Polypeptides

The invention also relates to variants, fragments, analogs and derivatives of the polypeptides described herein, including mutated PCTA-1 proteins.

The variant may be 1) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue and such substituted amino acid residue may or may not be one encoded by the genetic code, or 2) one in which one or more of the amino acid residues includes a substituent group, or 3) one in which the PCTA-1 protein is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or 4) one in which the additional amino acids are fused to the PCTA-1 protein, such as a leader or secretory sequence or a sequence which is employed for purification of the PCTA-1 protein or a preprotein sequence. Such variants are deemed to be within the scope of those skilled in the art

A polypeptide fragment is a polypeptide having a sequence that entirely is the same as part but not all of a given polypeptide sequence, preferably a polypeptide encoded by a PCTA-1 gene and variants thereof. Preferred fragments include those of the active region of the PCTA-1 protein that may play a role in prostate cancer and those regions possessing antigenic properties and which can be used to raise antibodies against the PCTA-1 protein.

In the case of an amino acid substitution in the amino acid sequence of a polypeptide according to the invention, one or several amino acids can be replaced by “equivalent” amino acids. The expression “equivalent” amino acid is used herein to designate any amino acid that may be substituted for one of the amino acids having similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Generally, the following groups of amino acids represent equivalent changes: (1) Ala, Pro, Gly, Glu, Asp, Gln, Asn, Ser, Thr; (2) Cys, Ser, Tyr, Thr; (3) Val, Ile, Leu, Met, Ala, Phe; (4) Lys, Arg, His; (5) Phe, Tyr, Trp, His.

A specific embodiment of a modified PCTA-1 peptide molecule of interest according to the present invention, includes, but is not limited to, a peptide molecule which is resistant to proteolysis, is a peptide in which the —CONH— peptide bond is modified and replaced by a (CH₂NH) reduced bond, a (NHCO) retro inverso bond, a (CH₂—O) methylene-oxy bond, a (CH₂—S) thiomethylene bond, a (CH2CH₂) carba bond, a (CO—CH₂) cetomethylene bond, a (CHOH—CH₂) hydroxyethylene bond), a (N—N) bound, a E-alcene bond or also a —CH═CH— bond. The invention also encompasses a human PCTA-1 polypeptide or a fragment or a variant thereof in which at least one peptide bound has been modified as described above.

Such fragments may be “free-standing”, i.e. not part of or fused to other polypeptides, or they may be comprised within a single larger polypeptide of which they form a part or region. However, several fragments may be comprised within a single larger polypeptide.

As representative examples of polypeptide fragments of the invention, there may be mentioned those which have from about 5, 6, 7, 8, 9 or 10 to 15, 10 to 20, 15 to 40, or 30 to 55 amino acids long. Preferred are those fragments containing at least one amino acid mutation in the PCTA-1 protein.

Identity between Nucleic Acids or Polypeptides

The terms “percentage of sequence identity” and “percentage homology” are used interchangeably herein to refer to comparisons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Homology is evaluated using any of the variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, 1988; Altschul et al., 1990; Thompson et al., 1994; Higgins et al., 1996; Altschul et al., 1993). In a particularly preferred embodiment, protein and nucleic acid sequence homologies are evaluated using the Basic Local Alignment Search Tool (“BLASr”) which is well known in the art (see, e.g., Karlin and Altschul, 1990; Altschul et al., 1990, 1993, 1997). In particular, five specific BLAST programs are used to perform the following task:

(1) BLASTP and BLAST3 compare an amino acid query sequence against a protein sequence database;

(2) BLASTN compares a nucleotide query sequence against a nucleotide sequence database;

(3) BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database;

(4) TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands); and

(5) TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.

The BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as “high-scoring segment pairs,” between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database. High-scoring segment pairs are preferably identified (i.e., aligned) by means of a scoring matrix, many of which are known in the art. Preferably, the scoring matrix used is the BLOSUM62 matrix (Gonnet et al., 1992; Henikoff and Henikoff, 1993). Less preferably, the PAM or PAM250 matrices may also be used (see, e.g., Schwartz and Dayhoff, eds., 1978). The BLAST programs evaluate the statistical significance of all high-scoring segment pairs identified, and preferably selects those segments which satisfy a user-specified threshold of significance, such as a user-specified percent homology. Preferably, the statistical significance of a high-scoring segment pair is evaluated using the statistical significance formula of Karlin (see, e.g., Karlin and Altschul, 1990).

Stringent Hybridization Conditions

By way of example and not limitation, procedures using conditions of high stringency are as follows: Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65° C. in buffer composed of 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 65° C., the preferred hybridization temperature, in prehybridization mixture containing 100 μg/ml denatured salmon sperm DNA and 5−20×10⁶ cpm of ³²P-labeled probe.

Alternatively, the hybridization step can be performed at 65° C. in the presence of SSC buffer, 1×SSC corresponding to 0.15M NaCl and 0.05 M Na citrate. Subsequently, filter washes can be done at 37° C. for 1 h in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA, followed by a wash in 0.1×SSC at 50° C. for 45 min. Alternatively, filter washes can be performed in a solution containing 2×SSC and 0.1% SDS, or 0.5×SSC and 0.1% SDS, or 0.1×SSC and 0.1% SDS at 68° C. for 15 minute intervals. Following the wash steps, the hybridized probes are detectable by autoradiography. Other conditions of high stringency which may be used are well known in the art and as cited in Sambrook et al., 1989; and Ausubel et al., 1989. These hybridization conditions are suitable for a nucleic acid molecule of about 20 nucleotides in length. There is no need to say that the hybridization conditions described above are to be adapted according to the length of the desired nucleic acid, following techniques well known to the one skilled in the art. The suitable hybridization conditions may for example be adapted according to the teachings disclosed in the book of Hames and Higgins (1985) or in Sambrook et al.(1989).

Genomic Sequence of the PCTA-1 Gene

The present invention relates to a purified and/or isolated nucleic acid corresponding to the genomic sequence of the PCTA-1 gene. Preferably, this genomic PCTA-1 sequence comprises the nucleotide sequence of SEQ ID No 1, a sequence complementary thereto, a fragment or a variant thereof.

The present invention encompasses the genomic sequence of PCTA-1. The PTCA-1 gene sequence comprises a coding sequence including 13 exons included in SEQ ID No 1, namely exon 0, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 6bis, exon 7, exon 9, exon 9bis and exon 9ter, the intronic regions, the promoter, the 5′UTR, the 3′UTR, and regulatory regions located upstream and downstream of the coding region.

The localization of the exons and introns of the PCTA-1 gene is detailed in Table A and is described as feature in SEQ ID No 1.

TABLE A Position Range Position range in SEQ ID No 1 in SEQ ID No 1 Exon Beginning End Intron Beginning End 0 68648 68741 0 68742 70646 1 70647 70794 1 70795 82207 2 82208 82296 2 82297 83612 3 83613 83823 3 83824 85297 4 85298 85417 4 85418 86388 5 86389 86445 5 86446 87495 6 87496 87522 6 87523 87649 6bis 87650 87775 6bis 87776 88294 7 88295 88383 7 88384 89483 8 89484 89649 8 89650 92748 9 92749 97155 9bis 92884 95820 9bis 92749 92883 9ter 95821 97155

Intron 0 refers to the nucleotide sequence located between Exon 0 and Exon 1, and so on. The intron 6 refers to the nucleotide sequence located between Exon 6 and Exon 6bis. The intron 6bis refers to the nucleotide sequence located between Exon 6bis and Exon 7. The intron 8 refers to the nucleotide sequence located between Exon 8 and Exon 9 or 9bis. The intron 9bis refers to the nucleotide sequence located between Exon 9bis and Exon 9ter.

The invention also encompasses a purified, isolated, or recombinant polynucleotide comprising a mucleotide sequence having at least 70, 75, 80, 85, 90, or 95% nucleotide identity with a mucleotide sequence of SEQ ID No 1 or a complementary sequence thereto or a fragment therof. The nucleotide differences as regards to the nucleotide sequence of SEQ ID No 1 may be generally randomly distributed throughout the entire nucleic acid. Nevertheless, preferred nucleic acids are those wherein the nucleotide differences as regards to the nucleotide sequence of SEQ ID No 1 are predominantly located outside the coding sequences contained in the exons. These nucleic acids, as well as their fragments and variants, may be used as oligonucleotide primers or probes in order to detect the presence of a copy of the PCTA-1 gene in a test sample, or alternatively in order to amplify a target nucleotide sequence within the PCTA-1 sequences.

Another object of the invention consists of a purified, isolated, or recombinant nucleic acid that hybridizes with the nucleotide sequence of SEQ ID No 1 or a complementary sequence therto or a variant thereof, under the stringent hybridization conditions as defined above.

Particularly preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1 or the complements therof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 1: 1-70715, 70795-82207, 82297-83612, 83824-85297, 85418-86388, 86446-87495, 87523-88294, 88384-89483, 89650-92748, 97156-98309, 98476-99329, 99491-100026, 100212-100281, 100396-100538, 100682-100833, 100995-101920, 102087-102970, 103264-103724, and 103753-106746. Additional preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide G at positions 70728, 87860, 88297, 94432, and 95340 of SEQ ID No 1; a nucleotide A at positions 82218, 83644, 83808, 87787, 87806, 94218, and 97144 of SEQ ID No 1; a nucleotide C at positions 87902, 88215, 88283, 92760, 93726, and 94422 of SEQ ID No 1; and a nucleotide T at positions 93903, and 94170 of SEQ ID No 1. Other preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide G at positions 86435, 93592, 93680, 93681, 93682,93728, 93761, and 95445 of SEQ ID No 1; a nucleotide A at positions 86434, 88355, 93240, 93471, and 93747of SEQ ID No 1; a nucleotide C at positions 93683, 95126, and 95444 of SEQ ID No 1; and a nucleotide T at positions 94154, and 94430 of SEQ ID No 1. Other preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises nucleotide positions selected from the group consisting of the nucleotide positions of SEQ ID No 1: 92975-92977, 93711-93715, 94151-94153, 94240-94243, 94770-94773, 94804-94808, 95121-95122, 95129-95135, 95148-95153, 95154-95159, 95173-95178, 95367-95374, 95410-95413, 95418-95420, 95430-95436, 95533-95535, and 95677-95677. It should be noted that nucleic acid fragments of any size and sequence may also be comprised by the polynucleotides described in this section.

A preferred aspect of the present invention is a purified and/or isolated and/or recombined PCTA-1 gene or a fragment thereof comprising at least one of the biallelic polymorphisms described below, a sequence complementary thereto, a fragment or a variant thereof. In some embodiments, the PCTA-1 gene or a fragment thereof may comprise at least one of the nucleotide sequences of P1 to P125, a sequence complementary thereto, a fragment or a variant thereof. In a preferred embodiment, the PCTA-1 gene or a fragment thereof comprises a biallelic marker selected from the group consisting of A1 to A125 and the complements thereof.

While this section is entitled “Genomic Sequences of The PCTA-1 Gene”, it should be noted that nucleic acid fragments of any size and sequence may also be comprised by the polynucleotides described in this section, flanking the genomic sequences of PCTA-1 on either side or between two or more such genomic sequences.

PCTA-1 cDNA Sequences

The invention also concerns a purified and/or isolated cDNA encoding a PCTA-1 protein; Preferably, the cDNA comprises a nucleotide sequence selected from the group consisting of SEQ ID Nos 2, 3, 4, sequences complementary thereto and functional fragments and variants thereof. Moreover, preferred polynucleotides of the invention include purified, isolated, or recombinant PCTA-1 cDNAs consisting of, consisting essentially of, or comprising a sequence selected from the group consisting of SEQ ID Nos 2, 3, 4 and the complementary sequence thereto.

The invention also pertains to a purified or isolated nucleic acid comprising a polynucleotide having at least 95% nucleotide identity with a polynucleotide selected from the group consisting of SEQ ID Nos 2, 3, 4, advantageously 99% nucleotide identity, preferably 0 99.5% nucleotide identity and most preferably 99.8% nucleotide identity with a polynucleotide selected from the group consisting of SEQ ID Nos 2, 3, 4, or a sequence complementary thereto or a biologically active fragment thereof.

Another object of the invention consists of purified, isolated or recombinant nucleic acids comprising a polynucleotide that hybridizes, under the stringent hybridization conditions defined herein, with a polynucleotide selected from the group consisting of the nucleotide sequences of SEQ ID Nos 2, 3, 4, or a sequence complementary thereto or a variant thereof or a biologically active fragment thereof.

The 5′UTR and 3′UTR regions of a gene are of particular importance in that they often comprise regulatory elements which can play a role in providing appropriate expression levels, particularly through the control of mRNA stability. The inventors have cloned a complete PCTA-1 cDNA (SEQ ID No 2) in which the 5′UTR is carried by exon 0 and a portion of exon 1 and the 3′UTR is carried by a portion of exon 9. Moreover, they have characterized a 5′EST, which is located as a feature in SEQ ID No 1, comprising the exons 0 and 1, and partially exon 2. Since an ATG codon is located at the beginning of the partial exon 1 disclosed in WO 96/21671, one could assume that the promoter of the PCTA-1 gene would be located immediately upstream of this codon. However, the inventors unexpectedly found that the PCTA-1 genomic DNA contains further exonic sequences upstream of the partial exon 1 disclosed in WO 96/21671. Without the knowledge of such sequences, the identification by the skilled person of the PCTA-1 promoter was extremely unlikely. Only the full genomic sequence of PCTA-1 and access by the inventors to a proprietary 5′EST database rendered possible the identification of a full cDNA sequence and of the PCTA-1 promoter. The invention concern the nucleotide sequence of 5′ EST consisting of the position range 1-266 in the SEQ ID No 2.

The main characteristics of the PCTA-1 cDNA comprising exons 0, 1, 2, 3, 4, 5, 6, 7, 8, and 9 are detailed in Table B. The invention concerns the purified and/or isolated sequence of the 5′UTR and 3′UTR as described in Table B or a complementary sequence thereto or an allelic variant thereof set forth in SEQ ID No 2. Particularly preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60,70, 80, 90, 100, 150,200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the nucleotide positions 1-162 of SEQ ID No 2. Further preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 253, 363, 527, 2471, and 5397 of SEQ ID No 2; a nucleotide C at positions 1013, 1979, and 2675 of SEQ ID No 2; a nucleotide G at positions 176, 749, 2685, 3593 of SEQ ID No 2; and a nucleotide T at positions 2156, and 2423 of SEQ ID No 2. Additional preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 708, 807, 1493, 1724, and 2000; a nucleotide C at positions 1936, 3379, and 3697; a nucleotide G at positions 709, 1845, 1933, 1934, 1935, 1981, 2014, and 3698; and a nucleotide T at positions 2407, and 2683 of SEQ ID No 2. Other preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises nucleotide positions selected from the group consisting of the nucleotide positions of SEQ ID No 2: 1229-1231, 1964-1968, 2404-2406, 2493-2496, 3023-3026, 3057-3061, 3374-3375, 3382-3388, 3401-3406, 3407-3412, 3426-3431, 3620-3627, 3663-3666, 3671-3673, 3683-3689, 3786-3788 and 3930-3932. It should be noted that nucleic acid fragments of any size and sequence may also be comprised by the polynucleotides described in this section.

The majority of interrupted genes are transcribed into an RNA that gives rise to a single type of spliced mRNA. But the RNAs of some genes follow patterns of alternative splicing, wherein a single gene gives rise to more than one mRNA species. In some cases, the ultimate pattern of expression is dictated by the primary transcript, because the use of different startpoints or termination sequences alters the splicing pattern. In other cases, a single primary transcript is spliced in more than one way, and internal exons are substituted, added or deleted. In some cases, the multiple products all are made in the same cell, but in others, the process is regulated so that particular splicing patterns occur only under particular conditions.

At least three PCTA-1 cDNAs are produced by alternative splicing. The inventors have identified a minor species of PCTA-1 cDNA, disclosed in SEQ ID No 3, and comprising an additional exon 6bis which encodes 42 additional amino acids. In a further embodiment, the present invention concerns the additional exon of the PCTA-1 gene located between exon 6 and exon 7, namely exon 6bis, detailed as a feature in SEQ ID No 1 and in Table A, a sequence complementary thereto, and a fragment or variant thereof. The present invention embodies a PCTA-1 cDNA comprising the exon 6bis disclosed in SEQ ID No 1.

The main characteristics of this second PCTA-1 cDNA comprising exons 0, 1, 2, 3, 4, 5, 6, 6bis, 7, 8, and 9 are detailed in Table B. The amino acid sequence of this new PCTA-1 protein is disclosed in SEQ ID No 6. Particularly preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 3: 1-162 and 747-872. Further preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 253, 363, 527, 2597, and 5523 of SEQ ID No 3; a nucleotide C at positions 1139, 2105, and 2801 of SEQ ID No 3; a nucleotide G at positions 176, 875, 2811, 3719 of SEQ ID No 3; and a nucleotide T at positions 2282, and 2549 of SEQ ID No 3. Additional preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 708, 807, 1619, 1850, and 2126; a nucleotide C at positions 2062, 3505, and 3823; a nucleotide G at positions 709, 1971, 2059, 2060, 2061, 2107, 2140, and 3824; and a nucleotide T at positions 2533, and 2809 of SEQ ID No 3. Other preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises nucleotide positions selected from the group consisting of the nucleotide positions of SEQ ID No 3: 1355-1357, 1892-1894,2090-2094, 2530-2532, 2619-2622, 3149-3152, 3183-3187, 3500-3501, 3508-3514, 3527-3532, 3533-3538, 3552-3557, 3746-3749, 3789-3792, 3797-3799, 3809-3815, 3912-3914 and 4056-4058. It should be noted that nucleic acid fragments of any size and sequence may also be comprised by the polynucleotides described in this section.

The inventors have also identified a species of PCTA-1 cDNA comprising alternative exons to exon 9 which are called exons 9bis and 9ter. Its sequence is disclosed in SEQ ID No 4. The exon 9bis and 9ter correspond respectively to the beginning and the ends of the exon 9. The polynucleotide of the exon 9 located between exons 9bis and 9ter is spliced or deleted. The combination of exons 9bis and 9ter extends the ORF of the PCTA-1 gene.

The main characteristics of this second PCTA-1 cDNA comprising exons 0, 1, 2, 3, 4, 5, 6, 7, 8, 9bis and 9ter are detailed in Table B. The amino acid sequence of the new PCTA-1 protein encoded by this cDNA is disclosed in SEQ ID No 7. Particularly preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 4 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the nucleotide positions 1-162 of SEQ ID No 4. Further preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 4 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 253, 363, 527 and 2460 of SEQ ID No 4; a nucleotide c at position 1013 of SEQ ID No 4 and a nucleotide G at positions 176, and 749 of SEQ ID No 4. Additionally preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 4 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 708 and 807 and a nucleotide G at position 709 of SEQ No 4. Other preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 4 or the complements thereof, wherein said contiguous span comprises the pairs of nucleotide positions 1136-1137 of SEQ ID No 4. It should be noted that nucleic acid fragments of any size and sequence may also be comprised by the polynucleotides described in this section.

The invention further embodies purified, isolated, or recombinant polynucleotides comprising a nucleotide sequence selected from the group consisting of the 13 exons of the PCTA-1 gene, or a sequence complementary thereto. The invention also deals with purified, isolated, or recombinant nucleic acids comprising a combination of at least two exons of the PCTA-1 gene, wherein the polynucleotides are arranged within the nucleic acid, from the 5′-end to the 3 ′-end of said nucleic acid, in the same order as in SEQ ID No 1. In this specific embodiment of a purified or isolated nucleic acid according to the invention, said nucleic acid preferably comprises the exon 0 at its 5′ end and the exon 9 or 9ter at its 3′ end.

The 3′UTR sequence of PCTA-1 appears to include several polyadenylation sites. These polyadenylation sites could have an influence on the stability of the mRNA resulting from the transcription of the PCTA-1 genomic DNA.

The invention also concerns a purified and/or isolated cDNA sequence encoding a mouse PCTA-1 protein, particularly a cDNA comprising the nucleotide sequence of SEQ ID No 8, a sequence complementary thereto or a fragment and variant thereof. The main characteristics of the murine cDNA are detailed in Table B. Moreover, preferred polynucleotides of the invention include purified, isolated, or recombinant PCTA-1 cDNAs consisting of, consisting essentially of, or comprising the sequence of SEQ ID No 8 and the complementary sequence thereto.

The invention also pertains to a purified or isolated nucleic acid comprising a polynucleotide having at least 95% nucleotide identity with a polynucleotide of SEQ ID No 8, advantageously 99% nucleotide identity, preferably 99.5% nucleotide identity and most preferably 99.8% nucleotide identity with a polynucleotide of SEQ ID No 8, or a sequence complementary thereto or a biologically active fragment thereof.

Another object of the invention consists of purified, isolated or recombinant nucleic acids comprising a polynucleotide that hybridizes, under the stringent hybridization conditions defined herein, with a polynucleotide of SEQ ID No 8, or a sequence complementary thereto or a variant thereof or a biologically active fragment thereof.

Particularly preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 8 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 8: 1-500, 501-1000, 1001-1500, and 1501-1738.

TABLE B Position range Position range of ORF Position range Position range of cDNA of 5′UTR ATG STOP of 3′UTR polydenylation sites SEQ ID No 2 1-200 201-203 1149-1151 1152-5408 1773-1778, 3624-3629, 3828-3833, 5119-5124, 5381-5386, 5386-5391 SEQ ID No 3 1-200 201-203 1275-1277 1278-5534 1899-1904, 3750-3755, 3954-3959, 5245-5250, 5507-5512, 5512-5517 SEQ ID No 4 1-200 201-203 1305-1307 1308-2471 2182-2187, 2444-2449, 2449-2454 SEQ ID No 8 1-120 121-123 1068-1070 1071-1738

While this section is entitled “PCTA-1 cDNA Sequences,” it should be noted that nucleic acid fragments of any size and sequence may also be comprised by the polynucleotides described in this section, flanking the genomic sequences of PCTA-1 on either side or between two or more such genomic sequences.

Coding Regions

The invention also concerns a nucleotide sequence encoding the human PCTA-1 protein selected from the group consisting of SEQ ID No 5, 6, 7, sequences complementary thereto and fragments and variants thereof. The present invention embodies isolated, purified, and recombinant polynucleotides which encode polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 or 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 5, wherein said contiguous span includes:

a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 203 in SEQ ID No 5; and/or

at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 183 in SEQ ID No 5.

The present invention also embodies isolated, purified, and recombinant polynucleotides which encode polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 or 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 6, wherein said contiguous span includes:

a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 245 in SEQ ID No 6; and/or

at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 225 in SEQ ID No 6; and/or

at least 1, 2, 3, 5 or 10 of the amino acid encoded by the exon 6bis, more particularly at least 1, 2, 3, 5 or 10 of the amino acid positions 183-224 of the SEQ ID No 6.

The present invention further embodies isolated, purified, and recombinant polynucleotides which encode polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 or 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 7, wherein said contiguous span includes:

a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 203 in SEQ ID No 7; and/or

at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 183 in SEQ ID No 7; and/or

at least 1, 2, 3, 5 or 1 0 of the amino acid encoded by the exons 9bis and 9ter, more particularly at least 1, 2, 3, 5 or 10 of the amino acid positions 313-368 of the SEQ ID No 7.

The invention also concerns a nucleotide sequence encoding the murine PCTA-1 protein of SEQ ID No 9, sequences complementary thereto and fragments and variants thereof More particularly, the present invention embodies isolated, purified, and recombinant polynucleotides which encode polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 or 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 9, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following amino acid positions of SEQ ID No 9: 1-50, 51-100, 101-150, 151-200, 201-250, and 251-316.

The above disclosed polynucleotide that contains the coding sequence of the PCTA-1 gene may be expressed in a desired host cell or a desired host organism, when this polynucleotide is placed under the control of suitable expression signals. The expression signals may be either the expression signals contained in the regulatory regions in the PCTA-1 gene of the invention or in contrast the signals may be exogenous regulatory nucleic sequences. Such a polynucleotide, when placed under the suitable expression signals, may also be inserted in a vector for its expression and/or amplification.

Regulator Sequences of the PCTA-1 Gene

The present invention also concerns the purified and/or isolated sequences of the upstream regulatory region (5′ regulatory region) of the PCTA-1 gene, sequences complementary thereto, and fragments or variants thereof, particularly the nucleotide sequence located between positions 1 and 68647 of SEQ ID No 1, as well as any sequence of 8 to 3000 consecutive nucleotides, preferably of 8 to 500 consecutive nucleotides, included therein. More particularly, the invention further includes specific elements within this regulatory region. These elements include a promoter region. The promoter region appears to be located in the 10 kb region, preferably in the 5 kb region, more preferably in the 2 kb region, still more preferably in the 1 kb region, and more particularly in the 500 bp, upstream of the first exon of the PCTA-1 gene. Preferably, the promoter region has a nucleotide sequence located between positions 66647 and 68647 of SEQ ID No 1 as well as any functional sequence of at least 8 consecutive nucleotide, preferably 8 to 400 consecutive nucleotides, more preferably of 8 to 300 nucleotides included therein, sequences complementary thereto and fragments and variants thereof. Further comments are provided below on this region which is of a particular importance in the present invention.

Also included in the invention are regulatory sequences downstream of the PCTA-1 coding sequence (3′ regulatory region) such as those included in the nucleotide sequence located between positions 97156 and 106746 of SEQ ID No 1, sequences complementary thereto and fragments and variants thereof.

In order to identify the relevant biologically active polynucleotide fragments or variants of the 5′ or 3′ regulatory region, the one skilled in the art will refer to the book of Sambrook et al. (Sambrook et al., 1989) which describes the use of a recombinant vector carrying a marker gene (i.e. beta galactosidase, chloramphenicol acetyl transferase, etc.) the expression of which will be detected when placed under the control of a biologically active polynucleotide fragments or variants of the 5′ or 3′ regulatory region. Genomic sequences located upstream of the first exon of the PCTA-1 gene are cloned into a suitable promoter reporter vector, such as the pSEAP-Basic, pSEAP-Enhancer, pβgal-Basic, pβgal-Enhancer, or pEGFP-1 Promoter Reporter vectors available from Clontech, or pGL2-basic or pGL3-basic promoterless luciferase reporter gene vector from Promega. Briefly, each of these promoter reporter vectors include multiple cloning sites positioned upstream of a reporter gene encoding a readily assayable protein such as secreted alkaline phosphatase, luciferase, beta galactosidase, or green fluorescent protein. The sequences upstream the PCTA-1 coding region are inserted into the cloning sites upstream of the reporter gene in both orientations and introduced into an appropriate host cell. The level of reporter protein is assayed and compared to the level obtained from a vector which lacks an insert in the cloning site. The presence of an elevated expression level in the vector containing the insert with respect to the control vector indicates the presence of a promoter in the insert. If necessary, the upstream sequences can be cloned into vectors which contain an enhancer for increasing transcription levels from weak promoter sequences. A significant level of expression above that observed with the vector lacking an insert indicates that a promoter sequence is present in the inserted upstream sequence.

Promoter sequences within the upstream genomic DNA may be further defined by constructing nested 5′ and/or 3′ deletions in the upstream DNA using conventional techniques such as Exonuclease III or appropriate restriction endonuclease digestion. The resulting deletion fragments can be inserted into the promoter reporter vector to determine whether the deletion has reduced or obliterated promoter activity. In this way, the boundaries of the promoters may be defined. If desired, potential individual regulatory sites within the promoter may be identified using site directed mutagenesis or linker scanning to obliterate potential transcription factor binding sites within the promoter individually or in combination. The effects of these mutations on transcription levels may be determined by inserting the mutations into cloning sites in promoter reporter vectors. This type of assay is well-known to those skilled in the art and is described in WO 97/17359, U.S. Pat. No. 5,374,544, EP 582,796, U.S. Pat. No. 5,698,389, U.S. Pat. No. 5,643,746, U.S. Pat. No. 5,502,176, and U.S. Pat. No. 5,266,488, the disclosures of which are incorporated herein by reference in their entireties.

The strength and the specificity of the promoter of the PCTA-1 gene can be assessed through the expression levels of a detectable polynucleotide operably linked to the PCTA-1 promoter in different types of cells and tissues. The detectable polynucleotide may be either a polynucleotide that specifically hybridizes with a predefined oligonucleotide probe, or a polynucleotide encoding a detectable protein, including a PCTA-1 polypeptide or a fragment or a variant thereof. This type of assay is well-known to those skilled in the art and is described in U.S. Pat. No. 5,502,176, and U.S. Pat. No. 5,266,488, the disclosures of which are incorporated herein by reference in their entireties. In one embodiment, the efficacy of the promoter of the PCTA-1 gene is assessed in normal and cancer cells. In a preferred embodiment, the efficacy of the promoter of the PCTA-1 gene is assessed in normal cells and in cancer cells which can present different degrees of malignancy, more preferably cells from prostate tissue. Some of the methods are discussed in more detail below.

Polynucleotides carrying the regulatory elements located at the 5′ end and at the 3′ end of the PCTA-1 coding region may be advantageously used to control the transcriptional and translational activity of an heterologous polynucleotide of interest.

Thus, the present invention also concerns a purified or isolated nucleic acid comprising a polynucleotide which is selected from the group consisting of the 5′ and 3′ regulatory regions, or a sequence complementary thereto or a biologically active fragment or variant thereof. “5′ regulatory region” refers to the nucleotide sequence located between positions 1 and 68647 of SEQ ID No 1. “3′ regulatory region” refers to the nucleotide sequence located between positions 97156 and 106746 of SEQ ID No 1.

The invention also pertains to a purified or isolated nucleic acid comprising a polynucleotide having at least 95% nucleotide identity with a polynucleotide selected from the group consisting of the 5′ and 3′ regulatory regions, advantageously 99% nucleotide identity, preferably 99.5% nucleotide identity and most preferably 99.8% nucleotide identity with a polynucleotide selected from the group consisting of the 5′ and 3′ regulatory regions, or a sequence complementary thereto or a biologically active fragment thereof.

Another object of the invention consists of purified, isolated or recombinant nucleic acids comprising a polynucleotide that hybridizes, under the stringent hybridization conditions defined herein, with a polynucleotide selected from the group consisting of the nucleotide sequences of the 5′- and 3′ regulatory regions, or a sequence complementary thereto or a variant thereof or a biologically active fragment thereof.

Preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 1: 1-4000, 4001-8000, 8001-12000, 12001-16000, 16001-20000, 20001-24000, 24001-28000, 28001-32000, 32001-36000, 36001-40000, 40001-44000, 4400-8000, 48001-52000, 52001-56000, 56001-60000, 60001-64000, 64001-68647. Particularly preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 1: 66647-68647.

“Biologically active” polynucleotide derivatives of SEQ ID No 1 are polynucleotides comprising or alternatively consisting of a fragment of said polynucleotide which is functional as a regulatory region for expressing a recombinant polypeptide or a recombinant polynucleotide in a recombinant cell host. It could act either as an enhancer or as a repressor.

For the purpose of the invention, a nucleic acid or polynucleotide is “functional” as a regulatory region for expressing a recombinant polypeptide or a recombinant polynucleotide if said regulatory polynucleotide contains nucleotide sequences which contain transcriptional and translational regulatory information, and such sequences are “operably linked” to nucleotide sequences which encode the desired polypeptide or the desired polynucleotide.

The regulatory polynucleotides of the invention may be prepared from the nucleotide sequence of SEQ ID No 1 by cleavage using suitable restriction enzymes, as described for example in the book of Sambrook et al.(1989). The regulatory polynucleotides may also be prepared by digestion of SEQ ID No 1 by an exonuclease enzyme, such as Bal3 1 (Wabiko et al., 1986). These regulatory polynucleotides can also be prepared by nucleic acid chemical synthesis, as described elsewhere in the specification.

A preferred 5′-regulatory polynucleotide of the invention includes the 5′-untranslated region (5′-UTR) of the PCTA-1 cDNA, or a biologically active fragment or variant thereof. A preferred 3′-regulatory,polynucleotide of the invention includes the 3′-untranslated region (3′-UTR) of the PCTA-1 cDNA, or a biologically active fragment or variant thereof.

A further object of the invention consists of a purified or isolated nucleic acid comprising:

a) a nucleic acid comprising a regulatory nucleotide sequence selected from the group consisting of:

(i) a nucleotide sequence comprising a polynucleotide of the 5′ regulatory region or a complementary sequence thereto;

(ii) a nucleotide sequence comprising a polynucleotide having at least 95% of nucleotide identity with the nucleotide sequence of the 5′ regulatory region or a complementary sequence thereto;

(iii) a nucleotide sequence comprising a polynucleotide that hybridizes under stringent hybridization conditions with the nucleotide sequence of the 5′ regulatory region or a complementary sequence thereto; and

(iv) a biologically active fragment or variant of the polynucleotides in (i), (ii) and (iii);

b) a polynucleotide encoding a desired polypeptide or a nucleic acid of interest, operably linked to the nucleic acid defined in (a) above; and

c) Optionally, a nucleic acid comprising a 3′-regulatory polynucleotide, preferably a 3′-regulatory polynucleotide of the PCTA-1 gene.

In a specific embodiment of the nucleic acid defined above, said nucleic acid includes the 5′-untranslated region (5′-UTR) of the PCTA-1 cDNA, or a biologically active fragment or variant thereof. In a second specific embodiment of the nucleic acid defined above, said nucleic acid includes the 3′-untranslated region (3′-UTR) of the PCTA-1 cDNA, or a biologically active fragment or variant thereof.

The regulatory polynucleotide of the 5′ regulatory region, or its biologically active fragments or variants, is operably linked at the 5′-end of the polynucleotide encoding the desired polypeptide or polynucleotide.

The regulatory polynucleotide of the 3′ regulatory region, or its biologically active fragments or variants, is advantageously operably linked at the 3′-end of the polynucleotide encoding the desired polypeptide or polynucleotide.

The desired polypeptide encoded by the above-described nucleic acid may be of various nature or origin, encompassing proteins of prokaryotic or eukaryotic origin. Among the polypeptides expressed under the control of a PCTA-1 regulatory region include bacterial, fungal or viral antigens. Also encompassed are eukaryotic proteins such as intracellular proteins, like “house keeping” proteins, membrane-bound proteins, like receptors, and secreted proteins like endogenous mediators such as cytokines. The desired polypeptide may be the PCTA-1 protein, especially the protein of a amino acid sequence selected from the group consisting of SEQ ID Nos 5, 6, 7, 9, or a fragment or a variant thereof.

The desired nucleic acids encoded by the above-described polynucleotide, usually an RNA molecule, may be complementary to a desired coding polynucleotide, for example to a PCTA-1 coding sequence, and thus useful as an antisense polynucleotide.

Such a polynucleotide may be included in a recombinant expression vector in order to express the desired polypeptide or the desired nucleic acid in host cell or in a host organism. Suitable recombinant vectors that contain a polynucleotide such as described herein are disclosed elsewhere in the specification.

Polynucleotide Constructs

The terms “polynucleotide construct” and “recombinant polynucleotide” are used interchangeably herein to refer to linear or circular, purified or isolated polynucleotides that have been artificially designed and which comprise at least two nucleotide sequences that are not found as contiguous nucleotide sequences in their initial natural environment.

DNA Construct that Enables Directing Temporal and Spatial PCTA-1 Gene Expression in Recombinant Cell Hosts and in Transgenic Animals.

In order to study the physiological and phenotypic consequences of a lack of synthesis of the PCTA-1 protein, both at the cell level and at the multi cellular organism level, the invention also encompasses DNA constructs and recombinant vectors enabling a conditional expression of a specific allele of the PCTA-1 genomic sequence or cDNA and also of a copy of this genomic sequence or cDNA harboring substitutions, deletions, or additions of one or more bases as regards to the PCTA-1 nucleotide sequence of SEQ ID Nos 1, 2, 3, 4, 8, or a fragment thereof, these base substitutions, deletions or additions being located either in an exon, an intron or a regulatory sequence, but preferably in the 5′-regulatory sequence or in an exon of the PCTA-1 genomic sequence or within a PCTA-1 cDNA of SEQ ID Nos 2, 3, 4, or 8. In a preferred embodiment, the PCTA-1 sequence comprises a biallelic marker of the present invention. In a preferred embodiment, the PCTA-1 sequence comprises a biallelic marker of the present invention, preferably one of the biallelic markers A1 to A125 and the complements thereof.

The present invention embodies recombinant vectors comprising any one of the polynucleotides described in the present invention. More particularly, the polynucleotide constructs according to the present invention can comprise any of the polynucleotides described in the “PCTA-1 cDNA Sequences” section, the “Coding Regions” section, and the “Oligonucleotide Probes And Primers” section.

A first preferred DNA construct is based on the tetracycline resistance operon tet from E. coli transposon Tn 10 for controlling the PCTA-1 gene expression, such as described by Gossen et al.(1992, 1995) and Furth et al.(1994). Such a DNA construct contains seven let operator sequences from Tn 10 (tetop) that are fused to either a minimal promoter or a 5′-regulatory sequence of the PCTA-1 gene, said minimal promoter or said PCTA-1 regulatory sequence being operably linked to a polynucleotide of interest that codes either for a sense or an antisense oligonucleotide or for a polypeptide, including a PCTA-1 polypeptide or a peptide fragment thereof. This DNA construct is functional as a conditional expression system for the nucleotide sequence of interest when the same cell also comprises a nucleotide sequence coding for either the wild type (tTA) or the mutant (rTA) repressor fused to the activating domain of viral protein VP16 of herpes simplex virus, placed under the control of a promoter, such as the HCMVIE1 enhancer/promoter or the MMTV-LTR. Indeed, a preferred DNA construct of the invention comprise both the polynucleotide containing the let operator sequences and the polynucleotide containing a sequence coding for the tTA or the rTA repressor.

In a specific embodiment, the conditional expression DNA construct contains the sequence encoding the mutant tetracycline repressor rTA, the expression of the polynucleotide of interest is silent in the absence of tetracycline and induced in its presence.

DNA Constructs Allowing Homologous Recombination: Replacement Vectors

A second preferred DNA construct will comprise, from 5′-end to 3′-end: (a) a first nucleotide sequence that is comprised in the PCTA-1 genomic sequence; (b) a nucleotide sequence comprising a positive selection marker, such as the marker for neomycine resistance (neo); and (c) a second nucleotide sequence that is comprised in the PCTA-1 genomic sequence, and is located on the genome downstream the first PCTA-1 nucleotide sequence (a).

In a preferred embodiment, this DNA construct also comprises a negative selection marker located upstream the nucleotide sequence (a) or downstream the nucleotide sequence (c). Preferably, the negative selection marker consists of the thymidine kinase (tk) gene (Thomas et al., 1986), the hygromycine beta gene (Te Riele et al., 1990), the hprt gene (Van der Lugt et al., 1991; Reid et al., 1990) or the Diphteria toxin A fragment (Dt-A) gene (Nada et al., 1993; Yagi et al. 1990). Preferably, the positive selection marker is located within a PCTA-1 exon sequence so as to interrupt the sequence encoding a PCTA-1 protein. These replacement vectors are described, for example, by Thomas et al.(1986; 1987), Mansour et al.(1988) and Koller et al.(1992).

The first and second nucleotide sequences (a) and (c) may be indifferently located within a PCTA-1 regulatory sequence, an intronic sequence, an exon sequence or a sequence containing both regulatory and/or intronic and/or exon sequences. The size of the nucleotide sequences (a) and (c) ranges from 1 to 50 kb, preferably from 1 to 10 kb, more preferably from 2 to 6 kb and most preferably from 2 to 4 kb.

DNA Constructs Allowing Homologous Recombination: Cre-LoxP System.

These new DNA constructs make use of the site specific recombination system of the P1 phage. The P1 phage possesses a recombinase called Cre which interacts specifically with a 34 base pairs loxP site. The loxP site is composed of two palindromic sequences of 13 bp separated by a 8 bp conserved sequence (Hoess et al., 1986). The recombination by the Cre enzyme between two loxP sites having an identical orientation leads to the deletion of the DNA fragment.

The Cre-loxP system used in combination with a homologous recombination technique has been first described by Gu et al.(1993, 1994). Briefly, a nucleotide sequence of interest to be inserted in a targeted location of the genome harbors at least two loxP sites in the same orientation and located at the respective ends of a nucleotide sequence to be excised from the recombinant genome. The excision event requires the presence of the recombinase (Cre) enzyme within the nucleus of the recombinant cell host. The recombinase enzyme may be brought at the desired time either by (a) incubating the recombinant cell hosts in a culture medium containing this enzyme, by injecting the Cre enzyme directly into the desired cell, such as described by Araki et al.(1995), or by lipofection of the enzyme into the cells, such as described by Baubonis et al.(1993); (b) transfecting the cell host with a vector comprising the Cre coding sequence operably linked to a promoter functional in the recombinant cell host, which promoter being optionally inducible, said vector being introduced in the recombinant cell host, such as described by Gu et al.(1993) and Sauer et al.(1988); (c) introducing in the genome of the cell host a polynucleotide comprising the Cre coding sequence operably linked to a promoter functional in the recombinant cell host, which promoter is optionally inducible, and said polynucleotide being inserted in the genome of the cell host either by a random insertion event or an homologous recombination event, such as described by Gu et al.(1994).

In a specific embodiment, the vector containing the sequence to be inserted in the PCTA-1 gene by homologous recombination is constructed in such a way that selectable markers are flanked by loxP sites of the same orientation, it is possible, by treatment by the Cre enzyme, to eliminate the selectable markers while leaving the PCTA-1 sequences of interest that have been inserted by an homologous recombination event. Again, two selectable markers are needed: a positive selection marker to select for the recombination event and a negative selection marker to select for the homologous recombination event. Vectors and methods using the Cre-toxP system are described by Zou et al.(1994).

Thus, a third preferred DNA construct of the invention comprises, from 5′-end to 3′-end: (a) a first nucleotide sequence that is comprised in the PCTA-1 genomic sequence; (b) a nucleotide sequence comprising a polynucleotide encoding a positive selection marker, said nucleotide sequence comprising additionally two sequences defining a site recognized by a recombinase, such as a loxP site, the two sites being placed in the same orientation; and (c) a second nucleotide sequence that is comprised in the PCTA-1 genomic sequence, and is located on the genome downstream of the first PCTA-1 nucleotide sequence (a).

The sequences defining a site recognized by a recombinase, such as a loxP site, are preferably located within the nucleotide sequence (b) at suitable locations bordering the nucleotide sequence for which the conditional excision is sought. In one specific embodiment, two loxP sites are located at each side of the positive selection marker sequence, in order to allow its excision at a desired time after the occurrence of the homologous recombination event.

In a preferred embodiment of a method using the third DNA construct described above, the excision of the polynucleotide fragment bordered by the two sites recognized by a recombinase, preferably two loxP sites, is performed at a desired time, due to the presence within the genome of the recombinant host cell of a sequence encoding the Cre enzyme operably linked to a promoter sequence, preferably an inducible promoter, more preferably a tissue-specific promoter sequence and most preferably a promoter sequence which is both inducible and tissue-specific, such as described by Gu et al.(1994).

The presence of the Cre enzyme within the genome of the recombinant cell host may result of the breeding of two transgenic animals, the first transgenic animal bearing the PCTA-1-derived sequence of interest containing the loxP sites as described above and the second transgenic animal bearing the Cre coding sequence operably linked to a suitable promoter sequence, such as described by Gu et al.(1994).

Spatio-temporal control of the Cre enzyme expression may also be achieved with an adenovirus based vector that contains the Cre gene thus allowing infection of cells, or in vivo infection of organs, for delivery of the Cre enzyme, such as described by Anton and Graham (1995) and Kanegae et al.(1995).

The DNA constructs described above may be used to introduce a desired nucleotide sequence of the invention, preferably a PCTA-1 genomic sequence or a PCTA-1 cDNA sequence, and most preferably an altered copy of a PCTA-1 genomic or cDNA sequence, within a predetermined location of the targeted genome, leading either to the generation of an altered copy of a targeted gene (knock-out homologous recombination) or to the replacement of a copy of the targeted gene by another copy sufficiently homologous to allow an homologous recombination event to occur (knock-in homologous recombination). In a specific embodiment, the DNA constructs described above may be used to introduce a PCTA-1 genomic sequence or a PCTA-1 cDNA sequence comprising at least one biallelic marker of the present invention, preferably at least one biallelic marker selected from the group consisting of A1 to A125 and the complements thereof.

Oligonucleotide Probes and Primers

Polynucleotides derived from the PCTA-1 gene are useful in order to detect the presence of at least a copy of a nucleotide sequence of SEQ ID No 1, or a fragment, complement, or variant thereof in a test sample.

Particularly preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 1: 1-70715, 70795-82207, 82297-83612, 83824-85297, 85418-86388, 86446-87495, 87523-88294, 88384-89483, 89650-92748, 97156-98309, 98476-99329, 99491-100026, 100212-100281, 100396-100538, 100682-100833, 100995-101920, 102087-102970, 103264-103724, and 103753-106746. Additional preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide G at positions 70728, 87860, 88297, 94432, and 95340 of SEQ ID No 1; a nucleotide A at positions 82218, 83644, 83808, 87787, 87806, 94218, and 97144 of SEQ ID No 1; a nucleotide C at positions 87902, 88215, 88283, 92760, 93726, and 94422 of SEQ ID No 1; and a nucleotide T at positions 93903, and 94170 of SEQ ID No 1. Other preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide G at positions 86435, 93592, 93680, 93681, 93682, 93728, 93761, and 95445 of SEQ ID No 1; a nucleotide A at positions 86434, 88355, 93240, 93471, and 93747of SEQ ID No l; a nucleotide C at positions 93683, 95126, and 95444 of SEQ ID No 1; and a nucleotide T at positions 94154, and 94430 of SEQ ID No 1. Other preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises nucleotide positions selected from the group consisting of the nucleotide positions of SEQ ID No 1: 92975-92977, 93711-93715, 94151-94153, 94240-94243, 94770-94773, 94804-94808, 95121-95122, 95129-95135, 95148-95153, 95154-95159, 95173-95178, 95367-95374, 95410-95413, 95418-95420, 95430-95436, 95533-95535, and 95677-95677.

Another object of the invention is a purified, isolated, or recombinant polynucleotide comprising the nucleotide sequence of SEQ ID No 2, complementary sequences thereto, as well as allelic variants, and fragments thereof. Particularly preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the nucleotide positions 1-162 of SEQ ID No 2. Additional preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 253, 363, 527, 2471, and 5397 of SEQ ID No 2; a nucleotide C at positions 1013, 1979, and 2675 of SEQ ID No 2; a nucleotide G at positions 176, 749, 2685, 3593 of SEQ ID No 2; and a nucleotide T at positions 2156, and 2423 of SEQ ID No 2. Particularly preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 708, 807, 1493, 1724, and 2000; a nucleotide C at positions 1936, 3379, and 3697; a nucleotide G at positions 709, 1845, 1933, 1934, 1935, 1981, 2014, and 3698; and a nucleotide T at positions 2407, and 2683 of SEQ ID No 2. Other preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises nucleotide positions selected from the group consisting of the nucleotide positions of SEQ ID No 2: 1229-1231, 1964-1968, 24042406, 2493-2496, 3023-3026, 3057-3061, 3374-3375, 3382-3388, 3401-3406, 3407-3412, 3426-3431, 3620-3627, 3663-3666, 3671-3673, 3683-3689, 3786-3788 and 3930-3932.

A further object of the invention is a purified, isolated, or recombinant polynucleotide comprising the nucleotide sequence of SEQ ID No 3, complementary sequences thereto, as well as allelic variants, and fragments thereof. Particularly preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 3: 1-162 and 747-872. Additional preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 253, 363, 527, 2597, and 5523 of SEQ ID No 3; a nucleotide C at positions 1139, 2105, and 2801 of SEQ ID No 3; a nucleotide G at positions 176, 875, 2811, 3719 of SEQ ID No 3; and a nucleotide T at positions 2282, and 2549 of SEQ ID No 3. Additional preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 708, 807, 1619, 1850, and 2126; a nucleotide C at positions 2062, 3505, and 3823; a nucleotide G at positions 709, 1971, 2059, 2060, 2061, 2107, 2140, and 3824; and a nucleotide Tat positions 2533, and 2809 of SEQ ID No 3. Other preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises nucleotide positions selected from the group consisting of the nucleotide positions of SEQ ID No 3: 1355-1357, 1892-1894, 2090-2094, 2530-2532, 2619-2622, 3149-3152, 3183-3187, 3500-3501, 3508-3514, 3527-3532, 3533-3538, 3552-3557, 3746-3749, 3789-3792, 3797-3799, 3809-3815, 3912-3914 and 4056-4058.

An additional object of the invention is a purified, isolated, or recombinant polynucleotide comprising the nucleotide sequence of SEQ ID No 4, complementary sequences thereto, as well as allelic variants, and fragments thereof. Particularly preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 4 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the nucleotide positions 1-162 of SEQ ID No 4. Additional preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 4 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 253, 363, 527 and 2460 of SEQ ID No 4; a nucleotide C at position 1013 of SEQ ID No 4; and a nucleotide G at positions 176 and 749 of SEQ ID No 4. Additionally preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 4 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 708 and 807 and a nucleotide G at position 709 of SEQ ID No 4. Other preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 4 or the complements thereof, wherein said contiguous span comprises the pairs of nucleotide positions 1136-1137 of SEQ ID No 4.

One more object of the invention is a purified, isolated, or recombinant polynucleotide comprising the nucleotide sequence of SEQ ID No 8, complementary sequences thereto, as well as allelic variants, and fragments thereof. Particularly preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 8 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 8: 1-500, 501-1000, 1001-1500, and 1501-1738.

Thus, the invention also relates to nucleic acid probes characterized in that they hybridize specifically, under the stringent hybridization conditions defined above, with a nucleic acid selected from the group consisting of the nucleotide sequences:

a) 1-70715, 70795-82207, 82297-83612, 83824-85297, 85418-86388, 86446-87495, 87523-88294, 88384-89483, 89650-92748, 97156-98309, 98476-99329, 99491-100026, 100212-100281, 100396-100538, 100682-100833, 100995-101920, 102087-102970, 103264-103724, and 103753-106746 of SEQ ID No 1 or a variant thereof or a sequence complementary thereto;

b) 1-162 of SEQ ID No 2 or a variant thereof or a sequence complementary thereto;

c) 1-162 and 747-872 of SEQ ID No 3 or a variant thereof or a sequence complementary thereto;

d) 1-162 of SEQ ID No 4 or a variant thereof or a sequence complementary thereto;

e) SEQ ID No 8 or a variant thereof or a sequence complementary thereto.

In a preferred embodiment, the oligonucleotides of the invention can hybridize with at least a portion of an intron or of the regulatory sequences of the PCTA-1 gene. Particularly preferred oligonucleotides of the invention hybridize with a sequence comprised in an intron or in the regulatory sequences of the PCTA-1 gene. In an other preferred embodiment, the oligonucleotides of the invention can hybridize with at least a portion of an exon selected from the group of exons 0, 1, 6bis, 9, and 9ter.

The present invention also concerns oligonucleotides and groups of oligonucleotides for the detection of alleles of biallelic markers of the PCTA-1 gene, preferably those associated with cancer, preferably with prostate cancer, with an early onset of prostate cancer, with a susceptibility to prostate cancer, with the level of aggressiveness of prostate cancer tumors, with a modified or forthcoming expression of the PCTA-1 gene, with a modified or forthcoming production of the PCTA-1 protein, or with the production of a modified PCTA-1 protein. These oligonucleotides are characterized in that they can hybridize with a PCTA-1 gene, preferably with a polymorphic PCTA-1 gene and more preferably with a region of a PCTA-1 gene comprising a polymorphic site containing a specific allele associated with prostate cancer, with the level of aggressiveness of prostate cancer tumors or with modifications in the regulation of expression of the PCTA-1 gene. These oligonucleotides are useful either as primers for use in various processes such as DNA amplification and microsequencing or as probes for DNA recognition in hybridization analyses.

Therefore, another preferred embodiment of a probe according to the invention consists of a nucleic acid comprising a biallelic marker selected from the group consisting of A1 to A125 or the complements thereof, for which the respective locations in the sequence listing are provided in Table 2. In some embodiments, the oligonucleotides comprise the polymorphic base of a sequence selected from P1 to P125, and the complementary sequences thereto. In other embodiments, the oligonucleotides have a 3′ terminus immediately adjacent to a polymorphic base in the PCTA-1 gene, such as a polymorphic base comprised in one of the sequences P1 to P125, and the complementary sequence thereto. In other embodiments, the oligonucleotide is capable of discriminating between different alleles of a biallelic marker in the PCTA-1 gene, including the biallelic markers A1 to A125 and the complements thereof.

In one embodiment the invention encompasses isolated, purified, and recombinant polynucleotides consisting of, or consisting essentially of a contiguous span of 8 to 50 nucleotides of any one of SEQ ID Nos 1, 2, 3, 4 and the complement thereof, wherein said span includes a PCTA-1-related biallelic marker in said sequence; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A44, A46 to A53, A57, A58, A62 to A76, A81, A82, A86 to A91, A107, A118, A123 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A45, A54, A60, A61, A77 to A80, A83 to A85, A93, A102 to A106, A109, A110, A114, and A122, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A55, A56, A59, A92, A94 to A101, A108, A111 to A113, A115 to A117, and A119 to A121, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said contiguous span is 18 to 47 nucleotides in length and said biallelic marker is within 4 nucleotides of the center of said polynucleotide; optionally, wherein said polynucleotide consists of said contiguous span and said contiguous span is 25 nucleotides in length and said biallelic marker is at the center of said polynucleotide; optionally, wherein said polynucleotide consists of said contiguous span and said contiguous span is 47 nucleotides in length and said biallelic marker is at the center of said polynucleotide; optionally, wherein the 3′ end of said contiguous span is present at the 3′ end of said polynucleotide; and optionally, wherein the 3′ end of said contiguous span is located at the 3′ end of said polynucleotide and said biallelic marker is present at the 3′ end of said polynucleotide. In a preferred embodiment, said probes comprises, consists of, or consists essentially of a sequence selected from the following sequences: P1 to P125 and the complementary sequences thereto.

In another embodiment the invention encompasses isolated, purified and recombinant polynucleotides comprising, consisting of, or consisting essentially of a contiguous span of 8 to 50 nucleotides of SEQ ID Nos 1, 2, 3, 4, or the complements thereof, wherein the 3′ end of said contiguous span is located at the 3′ end of said polynucleotide, and wherein the 3′ end of said polynucleotide is located within 20 nucleotides upstream of a PCTA-1-related biallelic marker in said sequence; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A44, A46 to A53, A57, A58, A62 to A76, A81, A82, A86 to A91, A107, A118, and A123 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A45, A54, A60, A61, A77 to A80, A83 to A85, A93, A102 to A106, A109, A110, A114, and A122, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A55, A56, A59, A92A94to A101, A108, A111 to A113, A115to A117, and A119 to A121, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein the 3′ end of said polynucleotide is located 1 nucleotide upstream of said PCTA-1-related biallelic marker in said sequence; and optionally, wherein said polynucleotide consists essentially of a sequence selected from the following sequences: D1 to D125 and E1 to E125.

In a further embodiment, the invention encompasses isolated, purified, or recombinant polynucleotides comprising, consisting of, or consisting essentially of a sequence selected from the following sequences: B1 to B47 and C1 to C47.

In an additional embodiment, the invention encompasses polynucleotides for use in hybridization assays, sequencing assays, and enzyme-based mismatch detection assays for determining the identity of the nucleotide at a PCTA-1-related biallelic marker in SEQ ID Nos 1, 2, 3, 4, or the complements thereof, as well as polynucleotides for use in amplifying segments of nucleotides comprising a PCTA-1-related biallelic marker in SEQ ID Nos 1, 2, 3, 4, or the complements thereof; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A44, A46 to A53, A57, A58, A62 to A76, A81, A82, A86 to A91, Al107, A118, and A123 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A45, A54, A60, A61, A77 to A80, A83 to A85, A93, A102 to A106, A109, A110, A114, and A122, and complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A55A56A59A92A94to A101, A108A111 to A113A115 to A117, and A119 to A121, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith.

The formation of stable hybrids depends on the melting temperature (Tm) of the DNA. The Tm depends on the length of the primer or probe, the ionic strength of the solution and the G+C content. The higher the G+C content of the primer or probe, the higher is the melting temperature because G:C pairs are held by three H bonds whereas A:T pairs have only two. The GC content in the probes of the invention usually ranges between 10 and 75%, preferably between 35 and 60%, and more preferably between 40 and 55%.

A probe or a primer according to the invention has between 8 and 1000 nucleotides in length, or is specified to be at least 12, 15, 18, 20, 25, 35, 40, 50, 60, 70, 80, 100, 250, 500 or 1000 nucleotides in length. More particularly, the length of these probes and primers can range from 8, 10, 15, 20, or 30 to 100 nucleotides, preferably from 10 to 50, more preferably from 15 to 30 nucleotides. Shorter probes and primers tend to lack specificity for a target nucleic acid sequence and generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. Longer probes and primers are expensive to produce and can sometimes self-hybridize to form hairpin structures. The appropriate length for primers and probes under a particular set of assay conditions may be empirically determined by one of skill in the art. A preferred probe or primer consists of a nucleic acid comprising a polynucleotide selected from the group of the nucleotide sequences of P1 to P125 and the complementary sequence thereto, B1 to B47, C1 to C47, D1 to D125, E1 to E125, for which the respective locations in the sequence listing are provided in Tables 1, 2, 3 and 4.

The primers and probes can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences and direct chemical synthesis by a method such as the phosphodiester method of Narang et al.(1979), the phosphodiester method of Brown et al.(1979), the diethylphosphoramidite method of Beaucage et al.(1981) and the solid support method described in EP0 707 592, the disclosure of which is incorporated herein by reference in its entirety.

Detection probes are generally nucleic acid sequences or uncharged nucleic acid analogs such as, for example peptide nucleic acids which are disclosed in International Patent Application WO 92/20702, morpholino analogs which are described in U.S. Pat. Nos. 5,185,444; 5,034,506and 5,142,047, the disclosures of which are incorporated herein by reference in their entireties. The probe may have to be rendered “non-extendable” in that additional dNTPs cannot be added to the probe. In and of themselves analogs usually are non-extendable and nucleic acid probes can be rendered non-extendable by modifying the 3′ end of the probe such that the hydroxyl group is no longer capable of participating in elongation. For example, the 3′ end of the probe can be functionalized with the capture or detection label to thereby consume or otherwise block the hydroxyl group. Alternatively, the 3′ hydroxyl group simply can be cleaved, replaced or modified, U.S. patent application Ser. No. 07/049,061 filed Apr. 19, 1993 describes modifications, which can be used to render a probe non-extendable.

Any of the polynucleotides of the present invention can be labeled, if desired, by incorporating any label known in the art to be detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive substances (including, ³²p, ³⁵S, ³H, ¹²⁵I), fluorescent dyes (including, 5-bromodesoxyuridin, fluorescein, acetylaminofluorene, digoxigenin) or biotin. Preferably, polynucleotides are labeled at their 3′ and 5′ ends. Examples of non-radioactive labeling of nucleic acid fragments are described in the French patent No. FR-7810975 or by Urdea et al (1988) or Sanchez-Pescador et al (1988), the disclosures of which are incorporated herein by reference in their entireties. In addition, the probes according to the present invention may have structural characteristics such that they allow the signal amplification, such structural characteristics being, for example, branched DNA probes as those described by Urdea et al. in 1991 or in the European patent No. EP0 225 807 (Chiron), the disclosures of which are incorporated herein by reference in their entireties.

A label can also be used to capture the primer, so as to facilitate the immobilization of either the primer or a primer extension product, such as amplified DNA, on a solid support. A capture label is attached to the primers or probes and can be a specific binding member which forms a binding pair with the solid's phase reagent's specific binding member (e.g. biotin and streptavidin). Therefore depending upon the type of label carried by a polynucleotide or a probe, it may be employed to capture or to detect the target DNA. Further, it will be understood that the polynucleotides, primers or probes provided herein, may, themselves, serve as the capture label. For example, in the case where a solid phase reagent's binding member is a nucleic acid sequence, it may be selected such that it binds a complementary portion of a primer or probe to thereby immobilize the primer or probe to the solid phase. In cases where a polynucleotide probe itself serves as the binding member, those skilled in the art will recognize that the probe will contain a sequence or “tail” that is not complementary to the target. In the case where a polynucleotide primer itself serves as the capture label, at least a portion of the primer will be free to hybridize with a nucleic acid on a solid phase. DNA Labeling techniques are well known to the skilled technician.

The probes of the present invention are useful for a number of purposes. They can be notably used in Southern hybridization to genomic DNA. The probes can also be used to detect PCR amplification products. They may also be used to detect mismatches in the PCTA-1 gene or mRNA using other techniques.

Any of the polynucleotides, primers and probes of the present invention can be conveniently immobilized on a solid support. Solid supports are known to those skilled in the art and include the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, nitrocellulose strips, membranes, microparticles such as latex particles, sheep (or other animal) red blood cells, duracytes and others. The solid support is not critical and can be selected by one skilled in the art. Thus, latex particles, microparticles, magnetic or non-magnetic beads, membranes, plastic tubes, walls of microtiter wells, glass or silicon chips, sheep (or other suitable animal's) red blood cells and duracytes are all suitable examples. Suitable methods for immobilizing nucleic acids on solid phases include ionic, hydrophobic, covalent interactions and the like. A solid support, as used herein, refers to any material which is insoluble, or can be made insoluble by a subsequent reaction. The solid support can be chosen for its intrinsic ability to attract and immobilize the capture reagent. Alternatively, the solid phase can retain an additional receptor which has the ability to attract and immobilize the capture reagent. The additional receptor can include a charged substance that is oppositely charged with respect to the capture reagent itself or to a charged substance conjugated to the capture reagent. As yet another alternative, the receptor molecule can be any specific binding member which is immobilized upon (attached to) the solid support and which has the ability to immobilize the capture reagent through a specific binding reaction. The receptor molecule enables the indirect binding of the capture reagent to a solid support material before the performance of the assay or during the performance of the assay. The solid phase thus can be a plastic, derivatized plastic, magnetic or non-magnetic metal, glass or silicon surface of a test tube, microtiter well, sheet, bead, microparticle, chip, sheep (or other suitable animal's) red blood cells, duracytes® and other configurations known to those of ordinary skill in the art. The polynucleotides of the invention can be attached to or immobilized on a solid support individually or in groups of at least 2, 5, 8, 10, 12, 15, 20, or 25 distinct polynucleotides of the invention to a single solid support. In addition, polynucleotides other than those of the invention may be attached to the same solid support as one or more polynucleotides of the invention.

Consequently, the invention also deals with a method for detecting the presence of a nucleic acid comprising a nucleotide sequence selected from a group consisting of SEQ ID Nos 1, 2, 3, 4, 8, a fragment or a variant thereof and a complementary sequence thereto in a sample, said method comprising the following steps of:

a) bringing into contact a nucleic acid probe or a plurality of nucleic acid probes which can hybridize with a nucleotide sequence included in a nucleic acid selected form the group consisting of the nucleotide sequences of SEQ ID Nos 1, 2, 3, 4, 8, a fragment or a variant thereof and a complementary sequence thereto and the sample to be assayed; and

b) detecting the hybrid complex formed between the probe and a nucleic acid in the sample.

The invention further concerns a kit for detecting the presence of a nucleic acid comprising a nucleotide sequence selected from a group consisting of SEQ ID Nos 1, 2, 3, 4, 8, a fragment or a variant thereof and a complementary sequence thereto in a sample, said kit comprising:

a) a nucleic acid probe or a plurality of nucleic acid probes which can hybridize with a nucleotide sequence included in a nucleic acid selected form the group consisting of the nucleotide sequences of SEQ ID Nos 1, 2, 3, 4, 8, a fragment or a variant thereof and a complementary sequence thereto; and

b) optionally, the reagents necessary for performing the hybridization reaction.

In a first preferred embodiment of this detection method and kit, said nucleic acid probe or the plurality of nucleic acid probes are labeled with a detectable molecule. In a second preferred embodiment of said method and kit, said nucleic acid probe or the plurality of nucleic acid probes has been immobilized on a substrate. In a third preferred embodiment, the nucleic acid probe or the plurality of nucleic acid probes comprise either a sequence which is selected from the group consisting of the nucleotide sequences of P1 to P125 and the complementary sequence thereto, B1 to B47, C1 to C47, D1 to D125, E1 to E125 or a biallelic marker selected from the group consisting of A1 to A125 and the complements thereto.

Oligonucleotide Arrays

A substrate comprising a plurality of oligonucleotide primers or probes of the invention may be used either for detecting or amplifying targeted sequences in the PCTA-1 gene and may also be used for detecting mutations in the coding or in the non-coding sequences of the PCTA-1 gene.

Any polynucleotide provided herein may be attached in overlapping areas or at random locations on the solid support. Alternatively the polynucleotides of the invention may be attached in an ordered array wherein each polynucleotide is attached to a distinct region of the solid support which does not overlap with the attachment site of any other polynucleotide. Preferably, such an ordered array of polynucleotides is designed to be “addressable” where the distinct locations are recorded and can be accessed as part of an assay procedure. Addressable polynucleotide arrays typically comprise a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations. The knowledge of the precise location of each polynucleotides location makes these “addressable” arrays particularly useful in hybridization assays. Any addressable array technology known in the art can be employed with the polynucleotides of the invention. One particular embodiment of these polynucleotide arrays is known as the Genechips™, and has been generally described in U.S. Pat. No. 5,143,854; PCT publications WO 90/15070 and 92/10092, the disclosures of which are incorporated herein by reference in their entireties. These arrays may generally be produced using mechanical synthesis methods or light directed synthesis methods which incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis (Fodor et al., 1991). The immobilization of arrays of oligonucleotides on solid supports has been rendered possible by the development of a technology generally identified as “Very Large Scale Immobilized Polymer Synthesis” (VLSIPS™) in which, typically, probes are immobilized in a high density array on a solid surface of a chip. Examples of VLSIPSυ technologies are provided in U.S. Pat. Nos. 5,143,854; and 5,412,087 and in PCT Publications WO 90/15070, WO 92/10092 and WO 95/11995, the disclosures of which are incorporated herein by reference in their entireties, which describe methods for forming oligonucleotide arrays through techniques such as light-directed synthesis techniques. In designing strategies aimed at providing arrays of nucleotides immobilized on solid supports, further presentation strategies were developed to order and display the oligonucleotide arrays on the chips in an attempt to maximize hybridization patterns and sequence information. Examples of such presentation strategies are disclosed in PCT Publications WO 94/12305, WO 94/11530, WO 97/29212 and WO 97/31256.

In another embodiment of the oligonucleotide arrays of the invention, an oligonucleotide probe matrix may advantageously be used to detect mutations occurring in the PCTA-1 gene and preferably in its regulatory region. For this particular purpose, probes are specifically designed to have a nucleotide sequence allowing their hybridization to the genes that carry known mutations (either by deletion, insertion or substitution of one or several nucleotides). By known mutations, it is meant, mutations on the PCTA-1 gene that have been identified according, for example to the technique used by Huang et al.(1996) or Samson et al.(1996).

Another technique that is used to detect mutations in the PCTA-1 gene is the use of a high-density DNA array. Each oligonucleotide probe constituting a unit element of the high density DNA array is designed to match a specific subsequence of the PCTA-1 genomic DNA or cDNA. Thus, an array consisting of oligonucleotides complementary to subsequences of the target gene sequence is used to determine the identity of the target sequence with the wild gene sequence, measure its amount, and detect differences between the target sequence and the reference wild gene sequence of the PCTA-1 gene. In one such design, termed 4L tiled array, is implemented a set of four probes (A, C, G, T), preferably 15-nucleotide oligomers. In each set of four probes, the perfect complement will hybridize more strongly than mismatched probes. Consequently, a nucleic acid target of length L is scanned for mutations with a tiled array containing 4L probes, the whole probe set containing all the possible mutations in the known wild reference sequence. The hybridization signals of the 15-mer probe set tiled array are perturbed by a single base change in the target sequence. As a consequence, there is a characteristic loss of signal or a “footprint” for the probes flanking a mutation position. This technique was described by Chee et al. in 1996.

Consequently, the invention concerns an array of nucleic acid molecules comprising at least one polynucleotide described above as probes and primers. Preferably, the invention concerns an array of nucleic acid comprising at least two polynucleotides described above as probes and primers.

A further object of the invention consists of an array of nucleic acid sequences comprising either at least one of the sequences selected from the group consisting of P1 to P125, B1 to B47, C1 to C47, D1 to D 125, E1 to E125, the sequences complementary thereto, a fragment thereof of at least 8, 10, 12, 15, 18, 20, 25, 30, or 40 consecutive nucleotides thereof, and at least one sequence comprising a biallelic marker selected from the group consisting of A1 to A125 and the complements thereto.

The invention also pertains to an array of nucleic acid sequences comprising either at least two of the sequences selected from the group consisting of P1 to P125, B1 to B47, C1 to C47, D1 to D 125, E1 to E125, the sequences complementary thereto, a fragment thereof of at least 8 consecutive nucleotides thereof, and at least two sequences comprising a biallelic marker selected from the group consisting of A1 to A125 and the complements thereof.

PCTA-1 Proteins and Polypeptide Fragments thereof

The term “PCTA-1 polypeptides” is used herein to embrace all of the proteins and polypeptides of the present invention. Also forming part of the invention are polypeptides encoded by the polynucleotides of the invention, as well as fusion polypeptides comprising such polypeptides.

The invention embodies PCTA-1 proteins from humans, including isolated or purified PCTA-1 proteins consisting, consisting essentially, or comprising the sequence of SEQ ID No 5. It should be noted the PCTA-1 proteins of the invention are based on the naturally-occurring variant of the amino acid sequence of human PCTA-1, wherein the valine residue of amino acid position 170 has been replaced with a serine residue and the glutamine residue of amino acid position 203 has been replaced with a lysine residue. This variant protein and the fragments thereof which contain a serine at the amino acid position 170 and a lysine at the amino acid position 203 of SEQ ID No 5 are collectively referred to herein as “170-Ser 203-Lys variants.” In another embodiment, the present invention concerns a purified and/or isolated nucleic acid encoding the PCTA-1 protein of SEQ ID No 5 or variant or fragment thereof.

The invention also concerns a purified and/or isolated PCTA-1 protein comprising a sequence selected from the group consisting of SEQ ID Nos 6, 7 and variants and functional fragments thereof. In another embodiment, the present invention concerns a purified and/or isolated nucleic acid encoding the PCTA-1 protein comprising a sequence selected from the group consisting of SEQ ID Nos 6, 7 or a variant or a fragment thereof.

The invention also encompasses the amino acid sequence of a murine PCTA-1 protein, such as that of SEQ ID No 9, fragments and variants thereof. The invention also concerns a nucleotide sequence encoding the murine PCTA-1 protein of SEQ ID No 9, sequences complementary thereto and fragments and variants thereof.

The invention also relates to modified human and mouse PCTA-1 proteins and to fragments and variants thereof. The term “modified PCTA-1 protein” is intended to designate a PCTA-1 protein which, when compared to a native PCTA-1 protein of SEQ ID No 5, 6, or 7, bears at least one amino acid substitution, deletion or addition. More particularly, preferred modified PCTA-1 proteins include the proteins bearing at least one of the following amino acid substitutions:

a substitution from F to Y at position 18, a substitution from R to C at position 35, a substitution from V to M at position 55 and a substitution from S to R at position 183 in SEQ ID No 5;

a substitution from F to Y at position 18, a substitution from R to C at position 35, a substitution from V to M at position 55, a substitution from D to Y at position 204 and a substitution from S to R at position 225 in SEQ ID No 6; and

a substitution from F to Y at position 18, a substitution from R to C at position 35, a substitution from V to M at position 55 and a substitution from S to R at position 183 in SEQ ID No 7.

Modified proteins bearing two or more of such substitutions also fall within the scope of the present invention. Other preferred embodiments include regions of the modified PCTA-1 proteins of the invention, and particularly those regions bearing at least one of the substitutions described above. Particularly preferred regions are those possessing antigenic properties and which can be used in vaccine agents or to raise antibodies against the PCTA-1 protein, and which most preferably comprise at least one of the particular substitutions referred to above.

The term “modified PCTA-1 protein” also designates a truncated PCTA-1 protein consisting of the amino acid sequence 1-211 of SEQ ID No 7.

The present invention embodies isolated, purified, and recombinant polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 5, wherein said contiguous span includes:

a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 203 in SEQ ID No 5; and/or

at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 183 in SEQ ID No 5.

The present invention embodies isolated, purified, and recombinant polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 6, wherein said contiguous span includes:

a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 245 in SEQ ID No 6; and/or

at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 225 in SEQ ID No 6; and/or

at least 1, 2, 3, 5 or 10 of the amino acid encoded by the exon 6bis, more particularly at least 1, 2, 3, 5 or 10 of the amino acid positions 183-224 of the SEQ ID No 6.

The present invention embodies isolated, purified, and recombinant polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 7, wherein said contiguous span includes:

a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 203 in SEQ ID No 7; and/or

at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 183 in SEQ ID No 7; and/or

at least 1, 2, 3, 5 or 10 of the amino acid encoded by the exons 9bis and 9ter, more particularly at least 1, 2, 3, 5 or 10 of the amino acid positions 313-368 of the SEQ ID No 7.

The invention also concerns the truncated PCTA-1 protein consisting essentially of or consisting of the amino acid positions 1-211 of SEQ ID No 7.

In other preferred embodiments the contiguous stretch of amino acids from SEQ ID Nos 5, 6, 7 comprises the site of a mutation or functional mutation, including a deletion, addition, swap or truncation of the amino acids in the PCTA-1 protein sequence.

The present invention embodies isolated, purified, and recombinant polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 9.

PCTA-1 proteins are preferably isolated from human or mammalian tissue samples or expressed from human or mammalian genes. The PCTA-1 polypeptides of the invention can be made using routine expression methods known in the art. The polynucleotide encoding the desired polypeptide, is ligated into an expression vector suitable for any convenient host. Both eukaryotic and prokaryotic host systems is used in forming recombinant polypeptides, and a summary of some of the more common systems. The polypeptide is then isolated from lysed cells or from the culture medium and purified to the extent needed for its intended use. Purification is by any technique known in the art, for example, differential extraction, salt fractionation, chromatography, centrifugation, and the like. See, for example, Methods in Enzymology for a variety of methods for purifying proteins.

In addition, shorter protein fragments is produced by chemical synthesis. Alternatively the proteins of the invention is extracted from cells or tissues of humans or non-human animals. Methods for purifying proteins are known in the art, and include the use of detergents or chaotropic agents to disrupt particles followed by differential extraction and separation of the polypeptides by ion exchange chromatography, affinity chromatography, sedimentation according to density, and gel electrophoresis.

Any PCTA-1 cDNA, including SEQ ID Nos 2, 3, 4, 8, is used to express PCTA-1 proteins and polypeptides. The nucleic acid encoding a PCTA-1 protein or polypeptide to be expressed is operably linked to a promoter in an expression vector using conventional cloning technology. The PCTA-1 insert in the expression vector may comprise the full coding sequence for a PCTA-1 protein or a fragment thereof. For example, the PCTA-1 derived insert may encode a polypeptide as described above.

The expression vector is any of the mammalian, yeast, insect or bacterial expression systems known in the art. Commercially available vectors and expression systems are available from a variety of suppliers including Genetics Institute (Cambridge, Mass.), Stratagene (La Jolla, Calif.), Promega (Madison, Wis.), and Invitrogen (San Diego, Calif.). If desired, to enhance expression and facilitate proper protein folding, the codon context and codon pairing of the sequence is optimized for the particular expression organism in which the expression vector is introduced, as explained by Hatfield, et al., U.S. Pat. No. 5,082,767, the disclosure of which is incorporated herein by reference in its entirety.

In one embodiment, the entire coding sequence of a PCTA-1 cDNA through the poly A signal of the cDNA are operably linked to a promoter in the expression vector. Alternatively, if the nucleic acid encoding a fragment of the PCTA-1 protein lacks a methionine to serve as the initiation site, an initiating methionine can be introduced next to the first codon of the nucleic acid using conventional techniques. Similarly, if the insert from a PCTA-1 cDNA lacks a poly A signal, this sequence can be added to the construct by, for example, splicing out the Poly A signal from pSG5 (Stratagene) using BglI and SalI restriction endonuclease enzymes and incorporating it into the mammalian expression vector pXT1 (Stratagene). pXT1 contains the LTRs and a portion of the gag gene from Moloney Murine Leukemia Virus. The position of the LTRs in the construct allow efficient stable transfection. The vector includes the Herpes Simplex Thymidine Kinase promoter and the selectable neomycin gene. The nucleic acid encoding a PCTA-1 protein or a fragment thereof is obtained by PCR from a bacterial vector containing a PCTA-1 cDNA selected from the group consisting of SEQ ID Nos 2, 3, 4, and 8 using oligonucleotide primers complementary to the PCTA-1 cDNA or fragment thereof and containing restriction endonuclease sequences for Pst I incorporated into the 5′ primer and BglII at the 5′ end of the corresponding cDNA3′ primer, taking care to ensure that the sequence encoding the PCTA-1 protein or a fragment thereof is positioned properly with respect to the poly A signal. The purified fragment obtained from the resulting PCR reaction is digested with PstI, blunt ended with an exonuclease, digested with BglII, purified and ligated to pXT1, now containing a poly A signal and digested with BglII.

The ligated product is transfected into mouse NIH 3T3 cells using Lipofectin (Life Technologies, Inc., Grand Island, N.Y.) under conditions outlined in the product specification. Positive transfectants are selected after growing the transfected cells in 600 ug/ml G418 (Sigma, St. Louis, Mo.).

Alternatively, the nucleic acids encoding the PCTA-1 protein or a fragment thereof is cloned into pED6dpc2 (Genetics Institute, Cambridge, Mass.). The resulting pED6dpc2 constructs is transfected into a suitable host cell, such as COS 1 cells. Methotrexate resistant cells are selected and expanded.

The above procedures may also be used to express a mutant PCTA-1 protein responsible for a detectable phenotype or a fragment thereof.

The expressed proteins is purified using conventional purification techniques such as ammonium sulfate precipitation or chromatographic separation based on size or charge. The protein encoded by the nucleic acid insert may also be purified using standard immunochromatography techniques. In such procedures, a solution containing the expressed PCTA-1 protein or fragment thereof, such as a cell extract, is applied to a column having antibodies against the PCTA-1 protein or fragment thereof is attached to the chromatography matrix. The expressed protein is allowed to bind the immunochromatography column. Thereafter, the column is washed to remove non-specifically bound proteins. The specifically bound expressed protein is then released from the column and recovered using standard techniques.

To confirm expression of a PCTA-1 protein or a fragment thereof, the proteins expressed from host cells containing an expression vector containing an insert encoding a PCTA-1 protein or a fragment thereof can be compared to the proteins expressed in host cells containing the expression vector without an insert. The presence of a band in samples from cells containing the expression vector with an insert which is absent in samples from cells containing the expression vector without an insert indicates that the PCTA-1 protein or a fragment thereof is being expressed. Generally,the bandwill have the mobility expected for the PCTA-1 protein or fragment thereof. However, the band may have a mobility different than that expected as a result of modifications such as glycosylation, ubiquitination, or enzymatic cleavage.

Antibodies capable of specifically recognizing the expressed PCTA-1 protein or a fragment thereof are described below.

If antibody production is not possible, the nucleic acids encoding the PCTA-1 protein or a fragment thereof is incorporated into expression vectors designed for use in purification schemes employing chimeric polypeptides. In such strategies the nucleic acid encoding the PCTA-1 protein or a fragment thereof is inserted in frame with the gene encoding the other half of the chimera. The other half of the chimera is β-globin or a nickel binding polypeptide encoding sequence. A chromatography matrix having antibody to β-globin or nickel attached thereto is then used to purify the chimeric protein. Protease cleavage sites is engineered between the β-globin gene or the nickel binding polypeptide and the PCTA-1 protein or fragment thereof. Thus, the two polypeptides of the chimera is separated from one another by protease digestion.

One useful expression vector for generating β-globin chimeric proteins is pSG5 (Stratagene), which encodes rabbit β-globin. Intron II of the rabbit β-globin gene facilitates splicing of the expressed transcript, and the polyadenylation signal incorporated into the construct increases the level of expression. These techniques are well known to those skilled in the art of molecular biology. Standard methods are published in methods texts such as Davis et al., (1986) and many of the methods are available from Stratagene, Life Technologies, Inc., or Promega. Polypeptide may additionally be produced from the construct using in vitro translation systems such as the In vitro Express™ Translation Kit (Stratagene).

Antibodies that Bind PCTA-1 Polypeptides of the Invention

Any PCTA-1 polypeptide or whole protein may be used to generate antibodies capable of specifically binding to an expressed PCTA-1 protein or fragments thereof as described.

One antibody composition of the invention is capable of specifically binding or specifically bind to the 170-Ser 203-Lys variant of the PCTA-1 protein of SEQ ID No 5. An other antibody composition of the invention is capable of specifically binding or specifically bind to the PCTA-1 protein selected from the group consisting of amino acid sequences of SEQ ID Nos 6, 7, 9. For an antibody composition to specifically bind to a first variant of PCTA-1, it must demonstrate at least a 5%, 10%, 15%, 20%, 25%, 50%, or 100% greater binding affinity for a full length first variant of the PCTA-1 protein than for a full length second variant of the PCTA-1 protein in an ELISA, RIA, or other antibody-based binding assay.

In a preferred embodiment, the invention concerns antibody compositions, either polyclonal or monoclonal, capable of selectively binding, or selectively bind to an epitope-containing any one of the following polypeptides:

a) a polypeptide comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 5, wherein said epitope comprises:

i) a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 203 in SEQ ID No 5; and/or

ii) at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 183 in SEQ ID No 5;

b) a polypeptide comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 6, wherein said epitope comprises:

i) a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 245 in SEQ ID No 6; and/or

ii) at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 225 in SEQ ID No 6; and/or

iii) at least 1, 2, 3, 5 or 10 of the amino acid encoded by the exon 6bis, more particularly at least 1, 2, 3, 5 or 10 of the amino acid positions 183-224 of the SEQ ID No6;

c) a polypeptide comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 7, wherein said epitope comprises:

i) a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 203 in SEQ ID No 7; and/or

ii) at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 183 in SEQ ID No 7; and/or

iii) at least 1, 2, 3, 5 or 10 of the amino acid encoded by the exons 9bis and 9ter, more particularly at least 1, 2, 3, 5or 10 of the amino acid positions 313-368 of the SEQ ID No 7; and

d) a polypeptide comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 9.

The invention also concerns a purified or isolated antibody capable of specifically binding to a mutated PCTA-1 protein or to a fragment or variant thereof comprising an epitope of the mutated PCTA-1 protein. In another preferred embodiment, the present invention concerns an antibody capable of binding to a polypeptide comprising at least 10 consecutive amino acids of a PCTA-1 protein and including at least one of the amino acids which can be encoded by the trait causing mutations.

In a preferred embodiment, the invention concerns the use of any one of the following polypeptides in the manufacture of antibodies:

a) a polypeptide comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 5, wherein said contigous span comprises:

i) a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 203 in SEQ ID No 5; and/or

ii) at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 183 in SEQ ID No 5;

b) a polypeptide comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 6, wherein said contigous span comprises:

i) a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 245 in SEQ ID No 6; and/or

i) at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 225 in SEQ ID No 6; and/or

ii) at least 1, 2, 3, 5 or 10 of the amino acid encoded by the exon 6bis, more particularly at least 1, 2, 3, 5 or 10 of the amino acid positions 183-224 of the SEQ ID No 6;

c) a polypeptide comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 7, wherein said contigous span comprises:

) a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 203 in SEQ ID No 7; and/or

i) at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 183 in SEQ ID No 7; and/or

ii) at least 1, 2, 3, 5 or 10 of the amino acid encoded by the exons 9bis and 9ter, more particularly at least 1, 2, 3, 5 or 10 of the amino acid positions 313-368 of the SEQ ID No 7; and

d) a polypeptide comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 9.

Non-human animals or mammals, whether wild-type or transgenic, which express a different species of PCTA-1 than the one to which antibody binding is desired, and animals which do not express PCTA-1 (i.e. a PCTA-1 knock out animal as described in herein) are particularly useful for preparing antibodies. PCTA-1 knock out animals will recognize all or most of the exposed regions of a PCTA-1 protein as foreign antigens, and therefore produce antibodies with a wider array of PCTA-1 epitopes. Moreover, smaller polypeptides with only 10 to 30 amino acids may be useful in obtaining specific binding to any one of the PCTA-1 proteins. In addition, the humoral immune system of animals which produce a species of PCTA-1 that resembles the antigenic sequence will preferentially recognize the differences between the animal's native PCTA-1 species and the antigen sequence, and produce antibodies to these unique sites in the antigen sequence. Such a technique will be particularly useful in obtaining antibodies that specifically bind to any one of the PCTA-1 proteins.

Antibody preparations prepared according to either protocol are useful in quantitative immunoassays which determine concentrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample. The antibodies may also be used in therapeutic compositions for killing cells expressing the protein or reducing the levels of the protein in the body.

The antibodies of the invention may be labeled by any one of the radioactive, fluorescent or enzymatic labels known in the art.

Consequently, the invention is also directed to a method for detecting specifically the presence of a PCTA-1 polypeptide according to the invention in a biological sample, said method comprising the following steps:

a) bringing into contact the biological sample with a polyclonal or monoclonal antibody that specifically binds a PCTA-1 polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID Nos 5, 6, 7, 9, or to a peptide fragment or variant thereof; and

b) detecting the antigen-antibody complex formed.

The invention also concerns a diagnostic kit for detecting in vitro the presence of a PCTA-1 polypeptide according to the present invention in a biological sample, wherein said kit comprises:

a) a polyclonal or monoclonal antibody that specifically binds a PCTA-1 polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID Nos 5, 6, 7, 9, or to a peptide fragment or variant thereof, optionally labeled;

b) a reagent allowing the detection of the antigen-antibody complexes formed, said reagent carrying optionally a label, or being able to be recognized itself by a labeled reagent, more particularly in the case when the above-mentioned monoclonal or polyclonal antibody is not labeled by itself.

PCTA-1-Related Biallelic Markers

Advantages of the Biallelic Markers of the Present Invention

The PCTA-1-related biallelic markers of the present invention offer a number of important advantages over other genetic markers such as RFLP (Restriction fragment length polymorphism) and VNTR (Variable Number of Tandem Repeats) markers.

The first generation of markers, were RFLPs, which are variations that modify the length of a restriction fragment. But methods used to identify and to type RFLPs are relatively wasteful of materials, effort, and time. The second generation of genetic markers were VNTRs, which can be categorized as either minisatellites or microsatellites. Minisatellites are tandemly repeated DNA sequences present in units of 5-50 repeats which are distributed along regions of the human chromosomes ranging from 0.1 to 20 kilobases in length. Since they present many possible alleles, their informative content is very high. Minisatellites are scored by performing Southern blots to identify the number of tandem repeats present in a nucleic acid sample from the individual being tested. However, there are only 10⁴ potential VNTRs that can be typed by Southern blotting. Moreover, both RFLP and VNTR markers are costly and time-consuming to develop and assay in large numbers.

Single nucleotide polymorphism or biallelic markers can be used in the same manner as RFLPs and VNTRs but offer several advantages. SNP are densely spaced in the human genome and represent the most frequent type of variation. An estimated number of more than 10⁷ sites are scattered along the 3×10⁹ base pairs of the human genome. Therefore, SNP occur at a greater frequency and with greater uniformity than RFLP or VNTR markers which means that there is a greater probability that such a marker will be found in close proximity to a genetic locus of interest. SNP are less variable than VNTR markers but are mutationally more stable.

Also, the different forms of a characterized single nucleotide polymorphism, such as the biallelic markers of the present invention, are often easier to distinguish and can therefore be typed easily on a routine basis. Biallelic markers have single nucleotide based alleles and they have only two common alleles, which allows highly parallel detection and automated scoring. The biallelic markers of the present invention offer the possibility of rapid, high throughput genotyping of a large number of individuals.

Biallelic markers are densely spaced in the genome, sufficiently informative and can be assayed in large numbers. The combined effects of these advantages make biallelic markers extremely valuable in genetic studies. Biallelic markers can be used in linkage studies in families, in allele sharing methods, in linkage disequilibrium studies in populations, in association studies of case-control populations or of trait positive and trait negative populations. An important aspect of the present invention is that biallelic markers allow association studies to be performed to identify genes involved in complex traits. Association studies examine the frequency of marker alleles in unrelated case- and control-populations and are generally employed in the detection of polygenic or sporadic traits. Association studies may be conducted within the general population and are not limited to studies performed on related individuals in affected families (linkage studies). Biallelic markers in different genes can be screened in parallel for direct association with disease or response to a treatment. This multiple gene approach is a powerful tool for a variety of human genetic studies as it provides the necessary statistical power to examine the synergistic effect of multiple genetic factors on a particular phenotype, drug response, sporadic trait, or disease state with a complex genetic etiology.

PCTA-1-Related Biallelic Markers and Polynucleotides Related Thereto

The invention also concerns a purified and/or isolated PCTA-1-related biallelic marker located in the sequence of the PCTA-1 gene, preferably a bialielic marker comprising an allele associated with prostate cancer, with an early onset of prostate cancer, with a response to a prophylactic or therapeutic agent administered for cancer treatment, particularly prostate cancer, with the level of aggressiveness of prostate cancer tumors, with a modified or forthcoming expression of the PCTA-1 gene, with a modified or forthcoming production of the PCTA-1 protein, or with the production of a modified PCTA-1 protein. The term PCTA-1-related biallelic marker includes the biallelic markers designated A1 to A125. The invention also concerns sets of these biallelic markers.

125 biallelic markers were identified. They include 3 deletions, 6 insertions and 2 variable motifs. 40 biallelic markers, namely A45, A54 to A56, A59 to A61, A75, A76, A85, A93 to A122, were located in exonic region. 39 biallelic markers, namely A44, A46 to A53, A57 to A58, A62 to A74, A77 to A84, A86 to A92, were localized in intronic region of the PCTA-1 gene. 3 biallelic markers A123, A124 and A125 were in the 3′ regulatory region. 43 biallelic markers, namely A1 to A43, were located in the 5′ regulatory region. More particularly, 16 of them, namely A28 to A43, were in the promoter of the PCTA-1 gene.

Among the exonic biallelic markers, 6 of them change the amino acid sequence of a PCTA-1 protein. First, the biallelic marker A54 encodes either a residue tyrosine or phenylalanine. The biallelic marker A56 encodes either a residue cysteine or arginine. The marker A60 encodes either a residue valine or methionine. The marker A75 encodes either a residue aspartic acid or tyrosine. The marker A76 encodes either a leucine residue or a STOP. Finally, the biallelic marker A85 encodes either a residue serine or arginine.

The invention also relates to a purified and/or isolated nucleotide sequence comprising a polymorphic base of a PCTA-1-related biallelic marker, preferably of a biallelic marker selected from the group consisting of A1 to A125, and the complements thereof. The sequence has between 8 and 1000 nucleotides in length, and preferably comprises at least 8, 10, 12, 15, 18, 20, 25, 35, 40, 50, 60, 70, 80, 100, 250, 500 or 1000 contiguous nucleotides, to the extent that such lengths are consistent with the specific sequence, of a nucleotide sequence selected from the group consisting of SEQ ID Nos 1, 2, 3, 4, or a variant thereof or a complementary sequence thereto. These nucleotide sequences comprise the polymorphic base of either allele 1 or allele 2 of the considered biallelic marker. Optionally, said biallelic marker may be within 6, 5, 4, 3, 2, or 1 nucleotides of the center of said polynucleotide or at the center of said polynucleotide. Optionally, the 3′ end of said contiguous span may be present at the 3′ end of said polynucleotide. Optionally, biallelic marker may be present at the 3′ end of said polynucleotide. Optionally, the 3′ end of said polynucleotide may be located within or at least 2, 4, 6, 8, 10, 12, 15, 18, 20, 25, 50, 100, 250, 500, or 1000 nucleotides upstream of a PCTA-1-related biallelic marker in said sequence. Optionally, the 3′ end of said polynucleotide may be located 1 nucleotide upstream of a PCTA-1-related biallelic marker in said sequence. Optionally, said polynucleotide may further comprise a label. Optionally, said polynucleotide can be attached to solid support. In a further embodiment, the polynucleotides defined above can be used alone or in any combination.

In a preferred embodiment, the sequences comprising a polymorphic base of one of the biallelic markers listed in Table 2 are selected from the group consisting of the nucleotide sequences that have a contiguous span of, that consist of, that are comprised in, or that comprises a polynucleotide having one of the sequences set forth as the amplicons listed in Table 1 or a variant thereof or a complementary sequence thereto.

The invention further concerns a nucleic acid encoding a PCTA-1 protein, wherein said nucleic acid comprises a polymorphic base of a biallelic marker selected from the group consisting of A1 to A125 and the complements thereof.

The invention also relates to a purified and/or isolated nucleotide sequence comprising a sequence defining a biallelic marker located in the sequence of the PCTA-1 gene. Preferably, the sequences defining a biallelic marker include the polymorphic base of one of the sequences P1 to P125 or the complementary sequence thereto. In some embodiments, the sequences defining a biallelic marker comprise one of the sequences selected from the group consisting of P1to P125, or a fragment or variant thereof or a complementary sequence thereto, said fragment comprising the polymorphic base.

The invention also concerns a set of the purified and/or isolated nucleotide sequences defined above. More particularly, the set of purified and/or isolated nucleotide sequences comprises a group of sequences defining a combination of biallelic markers located in the sequence of the PCTA-1 gene, preferably wherein alleles of said biallelic markers or the combinations thereof are associated with prostate cancer, with the level of aggressiveness of prostate cancer tumors, or with a level of expression of the PCTA-1 gene.

In a preferred embodiment, the invention relates to a set of purified and/or isolated nucleotide sequences, each sequence comprising a sequence defining a biallelic marker located in the sequence of the PCTA-1 gene, wherein the set is characterized in that between about 30 and 100%, preferably between about 40 and 60%, more preferably between 50 and 60%, of the sequences defining a biallelic marker are selected from the group consisting of P1 to P125, or a fragment or variant thereof or a complementary sequence thereto, said fragment comprising the polymorphic base.

More particularly, the invention concerns a set of purified and/or isolated nucleotide sequences, each sequence comprising a sequence defining a different bialielic marker located in the sequence of the PCTA-1 gene, said biallelic marker being either included in one of the nucleotide sequences of P1 to P125 or a complementary sequence thereto, or a biallelic marker preferably located in the sequence of the PCTA-1 gene, more preferably biallelic markers A1 to A125 and the complements thereof, and/or in linkage disequilibrium with one of the markers A1 to A125.

The invention also relates to a set of at least two, preferably four, five, six, seven, eight or more nucleotide sequences selected from the group consisting of P1 to P125, or a fragment or variant thereof or a complementary sequence thereto, said fragment comprising the polymorphic base.

The invention further concerns a nucleotide sequence selected from the group consisting of P1 to P125 or a fragment or a variant thereof or a complementary sequence thereto, said fragment comprising the polymorphic base.

The invention also encompasses the use of any polynucleotide for, or any polynucleotide for use in, determining the identity of one or more nucleotides at a PCTA-1-related biallelic marker. In addition, the polynucleotides of the invention for use in determining the identity of one or more nucleotides at a PCTA-1-related biallelic marker encompass polynucleotides with any further limitation described in this disclosure, or those following, specified alone or in any combination. Optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A44, A46 to A53, A57, A58, A62 to A76, A81, A82, A86 to A91, A107, A118, and A123 complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A45, A54, A60, A61, A77 to A80, A83 to A85, A93, A102 to A106, A109, A110, A114, A122, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A55, A56, A59, A92, A94 to A101, A108, A111 to 113, A115, A117, and A119 to A121, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; Optionally, said polynucleotide may comprise a sequence disclosed in the present specification; Optionally, said polynucleotide may consist of, or consist essentially of any polynucleotide described in the present specification; Optionally, said determining may be performed in a hybridization assay, a sequencing assay, a microsequencing assay, or an enzyme-based mismatch detection assay; A preferred polynucleotide may be used in a hybridization assay for determining the identity of the nucleotide at a PCTA-1-related biallelic marker. Another preferred polynucleotide may be used in a sequencing or microsequencing assay for determining the identity of the nucleotide at a PCTA-1-related biallelic marker. A third preferred polynucleotide may be used in an enzyme-based mismatch detection assay for determining the identity of the nucleotide at a PCTA-1-related biallelic marker. A fourth preferred polynucleotide may be used in amplifying a segment of polynucleotides comprising a PCTA-1-related biallelic marker. Optionally, any of the polynucleotides described above may be attached to a solid support, array, or addressable array; Optionally, said polynucleotide may be labeled.

Additionally, the invention encompasses the use of any polynucleotide for, or any polynucleotide for use in, amplifying a segment of nucleotides comprising a PCTA-1-related biallelic marker. In addition, the polynucleotides of the invention for use in amplifying a segment of nucleotides comprising a PCTA-1-related biallelic marker encompass polynucleotides with any further limitation described in this disclosure, or those following, specified alone or in any combination: Optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A44, A46 to A53, A57, A58, A62 to A76, A81, A82, A86 to A91, A107, A118, and A123 complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A45, A54, A60, A61, A77 to A80, A83 to A85, A93, A102 to A106, A109, A122, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A55, A56, A59, A92, A94 to A101, A108, A111 to A113, A115 to A117, and A119 to A121, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; Optionally, said polynucleotide may comprise a sequence disclosed in the present specification; Optionally, said polynucleotide may consist of, or consist essentially of any polynucleotide described in the present specification; Optionally, said amplifying may be performed by a PCR or LCR. Optionally, said polynucleotide may be attached to a solid support, array, or addressable array. Optionally, said polynucleotide may be labeled.

The primers for amplification or sequencing reaction of a polynucleotide comprising a biallelic marker of the invention may be designed from the disclosed sequences for any method known in the art. A preferred set of primers are fashioned such that the 3′ end of the contiguous span of identity with a sequence selected from the group consisting of SEQ ID Nos 1, 2, 3, 4 or a sequence complementary thereto or a variant thereof is present at the 3′ end of the primer. Such a configuration allows the 3′ end of the primer to hybridize to a selected nucleic acid sequence and dramatically increases the efficiency of the primer for amplification or sequencing reactions. Allele specific primers may be designed such that a polymorphic base of a biallelic marker is at the 3′ end of the contiguous span and the contiguous span is present at the 3′ end of the primer. Such allele specific primers tend to selectively prime an amplification or sequencing reaction so long as they are used with a nucleic acid sample that contains one of the two alleles present at a biallelic marker. The 3′ end of the primer of the invention may be located within or at least 2, 4, 6, 8, 10, 12, 15, 18, 20, 25, 50, 100, 250, 500, or 1000 nucleotides upstream of a PCTA-1-related biallelic marker in said sequence or at any other location which is appropriate for their intended use in sequencing, amplification or the location of novel sequences or markers. Thus, another set of preferred amplification primers comprise an isolated polynucleotide consisting essentially of a contiguous span of 8 to 50 nucleotides in a sequence selected from the group consisting of SEQ ID Nos 1, 2, 3, 4 or a sequence complementary thereto or a variant thereof, wherein the 3′ end of said contiguous span is located at the 3′ end of said polynucleotide, and wherein the 3′ end of said polynucleotide is located upstream of a PCTA-1-related biallelic marker in said sequence. Preferably, those amplification primers comprise a sequence selected from the group consisting of the sequences B1 to B47 and C1 to C47. Primers with their 3′ ends located 1 nucleotide upstream of a biallelic marker of PCTA-1 have a special utility as microsequencing assays. Preferred microsequencing primers arc described in Table 4. Optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A44, A46 to A53, A57, A58, A62 to A76, A8 1, A82, A86 to A91, A107, A118, and A123 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A45, A54, A60, A61, A77 to A80, A83 to A85, A93, A102 to A106, A109, A110, A114, and A122, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A55, A56, A59, A92, A94to A101, A108, A111 to A113, A115 to A117 and A119 to A121, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; Optionally, microsequencing primers are selected from the group consisting of the nucleotide sequences D1 to D125 and E1 to E125. More preferred microsequencing primers are Q selected from the group consisting of the nucleotides sequences D15, D24, D30, D34, D36, D38, D41, D44, D50, D53, D54, D56, D57, D59, D76, D85, D93, D108, D111, D115, D124, E11, E14, E22, E25, E26, E35, E42, E52, E53, E55, E56, E60, E61, E64, E73, E75, E93, E96.

The probes of the present invention may be designed from the disclosed sequences for any method known in the art, particularly methods which allow for testing if a marker disclosed herein is present. A preferred set of probes may be designed for use in the hybridization assays of the invention in any manner known in the art such that they selectively bind to one allele of a biallelic marker, but not the other under any particular set of assay conditions. Preferred hybridization probes comprise the polymorphic base of either allele I or allele 2 of the considered biallelic marker. Optionally, said biallelic marker may be within 6, 5, 4, 3, 2, or 1 nucleotides of the center of the hybridization probe or at the center of said probe. In a preferred embodiment, the probes are selected from the group consisting of the sequences P1 to P125 and the complementary sequence thereto.

It should be noted that the polynucleotides of the present invention are not limited to having the exact flanking sequences surrounding the polymorphic bases which are enumerated in Sequence Listing. Rather, it will be appreciated that the flanking sequences surrounding the biallelic markers may be lengthened or shortened to any extent compatible with their intended use and the present invention specifically contemplates such sequences. The flanking regions outside of the contiguous span need not be homologous to native flanking sequences which actually occur in human subjects. The addition of any nucleotide sequence which is compatible with the nucleotides intended use is specifically contemplated.

Primers and probes may be labeled or immobilized on a solid support as described in“Oligonucleotide probes and primers”.

The polynucleotides of the invention which are attached to a solid support encompass polynucleotides with any further limitation described in this disclosure, or those following, specified alone or in any combination: Optionally, said polynucleotides may be specified as attached individually or in groups of at least 2, 5, 8, 10, 12, 15, 20, or 25 distinct polynucleotides of the invention to a single solid support. Optionally, polynucleotides other than those of the invention may attached to the same solid support as polynucleotides of the invention. Optionally, when multiple polynucleotides are attached to a solid support they may be attached at random locations, or in an ordered array. Optionally, said ordered array may be addressable.

The present invention also encompasses diagnostic kits comprising one or more polynucleotides of the invention with a portion or all of the necessary reagents and instructions for genotyping a test subject by determining the identity of a nucleotide at a PCTA-1-related biallelic marker. The polynucleotides of a kit may optionally be attached to a solid support, or be part of an array or addressable array of polynucleotides. The kit may provide for the determination of the identity of the nucleotide at a marker position by any method known in the art including, but not limited to, a sequencing assay method, a microsequencing assay method, a hybridization assay method, or an enzyme-based mismatch detection assay method.

Methods for De Novo Identification of Biallelic Markers

Any of a variety of methods can be used to screen a genomic fragment for single nucleotide polymorphisms such as differential hybridization with oligonucleotide probes, detection of changes in the mobility measured by gel electrophoresis or direct sequencing of the amplified nucleic acid. A preferred method for identifying biallelic markers involves comparative sequencing of genomic DNA fragments from an appropriate number of unrelated individuals.

In a first embodiment, DNA samples from unrelated individuals are pooled together, following which the genomic DNA of interest is amplified and sequenced. The nucleotide sequences thus obtained are then analyzed to identify significant polymorphisms. One of the major advantages of this method resides in the fact that the pooling of the DNA samples substantially reduces the number of DNA amplification reactions and sequencing reactions, which must be carried out. Moreover, this method is sufficiently sensitive so that a biallelic marker obtained thereby usually demonstrates a sufficient frequency of its less common allele to be useful in conducting association studies.

In a second embodiment, the DNA samples are not pooled and are therefore amplified and sequenced individually. This method is usually preferred when biallelic markers need to be identified in order to perform association studies within candidate genes. Preferably, highly relevant gene regions such as promoter regions or exon regions may be screened for biallelic markers. A biallelic marker obtained using this method may show a lower degree of informativeness for conducting association studies, e.g. if the frequency of its less frequent allele may be less than about 10%. Such a biallelic marker will, however, be sufficiently informative to conduct association studies and it will further be appreciated that including less informative biallelic markers in the genetic analysis studies of the present invention, may allow in some cases the direct identification of causal mutations, which may, depending on their penetrance, be rare mutations.

The following is a description of the various parameters of a preferred method used by the inventors for the identification of the biallelic markers of the present invention.

Genomic DNA Samples

The genomic DNA samples from which the biallelic markers of the present invention are generated are preferably obtained from unrelated individuals corresponding to a heterogeneous population of known ethnic background. The number of individuals from whom DNA samples are obtained can vary substantially, preferably from about 10 to about 1000, preferably from about 50 to about 200 individuals. It is usually preferred to collect DNA samples from at least about 100 individuals in order to have sufficient polymorphic diversity in a given population to identify as many markers as possible and to generate statistically significant results.

As for the source of the genomic DNA to be subjected to analysis, any test sample can be foreseen without any particular limitation. These test samples include biological samples, which can be tested by the methods of the present invention described herein, and include human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external secretions of the respiratory, intestinal and genitourinary tracts, tears, saliva, milk, white blood cells, myelomas and the like; biological fluids such as cell culture supernatants; fixed tissue specimens including tumor and non-tumor tissue and lymph node tissues; bone marrow aspirates and fixed cell specimens. The preferred source of genomic DNA used in the present invention is from peripheral venous blood of each donor. Techniques to prepare genomic DNA from biological samples are well known to the skilled technician. Details of a preferred embodiment are provided in Example 1. The person skilled in the art can choose to amplify pooled or unpooled DNA samples.

DNA Amplification

The identification of biallelic markers in a sample of genomic DNA may be facilitated through the use of DNA amplification methods. DNA samples can be pooled or unpooled for the amplification step. DNA amplification techniques are well known to those skilled in the art.

Amplification techniques that can be used in the context of the present invention include, but are not limited to, the ligase chain reaction (LCR) described in EP-A-320 308, WO 9320227 and EP-A439 182, the disclosures of which are incorporated herein by reference in their entireties, the polymerase chain reaction (PCR, RT-PCR) and techniques such as the nucleic acid sequence based amplification (NASBA) described in Guatelli J. C., et al.(1990) and in Compton J.(1991), Q-beta amplification as described in European Patent Application No 4544610, the disclosures of which are incorporated herein by reference in their entireties, strand displacement amplification as described in Walkeret al.(1996) and EP A684 315, the disclosures of which are incorporated herein by reference in their entireties, and, target mediated amplification as described in PCT Publication WO 9322461.

LCR and Gap LCR are exponential amplification techniques, both depend on DNA ligase to join adjacent primers annealed to a DNA molecule. In Ligase Chain Reaction (LCR), probe pairs are used which include two primary (first and second) and two secondary (third and fourth) probes, all of which are employed in molar excess to target. The first probe hybridizes to a first segment of the target strand and the second probe hybridizes to a second segment of the target strand, the first and second segments being contiguous so that the primary probes abut one another in 5′ phosphate-3′ hydroxyl relationship, and so that a ligase can covalently fuse or ligate the two probes into a fused product. In addition, a third (secondary) probe can hybridize to a portion of the first probe and a fourth (secondary) probe can hybridize to a portion of the second probe in a similar abutting fashion. Of course, if the target is initially double stranded, the secondary probes also will hybridize to the target complement in the first instance. Once the ligated strand of primary probes is separated from the target strand, it will hybridize with the third and fourth probes, which can be ligated to form a complementary, secondary ligated product. It is important to realize that the ligated products are functionally equivalent to either the target or its complement. By repeated cycles of hybridization and ligation, amplification of the target sequence is achieved. A method for multiplex LCR has also been described (WO 9320227, the disclosure of which is incorporated herein by reference in its entirety). Gap LCR (GLCR) is a version of LCR where the probes are not adjacent but are separated by 2 to 3 bases.

For amplification of mRNAs, it is within the scope of the present invention to reverse transcribe mRNA into cDNA followed by polymerase chain reaction (RT-PCR); or, to use a single enzyme for both steps as described in U.S. Pat. No. 5,322,770, the disclosure of which is incorporated herein by reference in its entirety, or, to use Asymmetric Gap LCR (RT-AGLCR) as described by Marshall et al.(1994). AGLCR is a modification of GLCR that allows the amplification of RNA.

The PCR technology is the preferred amplification technique used in the present invention. A variety of PCR techniques are familiar to those skilled in the art. For a review of PCR technology, see White (1997) and the publication entitled “PCR Methods and Applications” (1991, Cold Spring Harbor Laboratory Press). In each of these PCR procedures, PCR primers on either side of the nucleic acid sequences to be amplified are added to a suitably prepared nucleic acid sample along with dNTPs and a thermostable polymerase such as Taq polymerase, Pfu polymerase, or Vent polymerase. The nucleic acid in the sample is denatured and the PCR primers are specifically hybridized to complementary nucleic acid sequences in the sample. The hybridized primers are extended. Thereafter, another cycle of denaturation, hybridization, and extension is initiated. The cycles are repeated multiple times to produce an amplified fragment containing the nucleic acid sequence between the primer sites. PCR has further been described in several patents including U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,965,188, the disclosures of which are incorporated herein by reference in their entireties.

The PCR technology is the preferred amplification technique used to identify new biallelic markers. A typical example of a PCR reaction suitable for the purposes of the present invention is provided in Example 2.

One of the aspects of the present invention is a method for the amplification of the human PCTA-1 gene, particularly of a fragment of the genomic sequence of SEQ ID No 1 or of the cDNA sequences of SEQ ID Nos 2, 3, 4, 8, or a fragment or a variant thereof in a test sample, preferably using the PCR technology. This method comprises the steps of:

a) contacting a test sample with amplification reaction reagents comprising a pair of amplification primers as described above and located on either side of the polynucleotide region to be amplified, and

b) optionally, detecting the amplification products.

The invention also concerns a kit for the amplification of a PCTA-1 gene sequence, particularly of a portion of the genomic sequence of SEQ ID No 1 or of the cDNA sequences of SEQ ID Nos 2, 3 4, 9, or a variant thereof in a test sample, wherein said kit comprises:

a) a pair of oligonucleotide primers located on either side of the PCTA-1 region to be amplified;

b) optionally, the reagents necessary for performing the amplification reaction.

In one embodiment of the above amplification method and kit, the amplification product is detected by hybridization with a labeled probe having a sequence which is complementary to the amplified region. In another embodiment of the above amplification method and kit, primers comprise a sequence which is selected from the group consisting of the nucleotide sequences of B1 to B47, C1 to C47, D1 to D125, and E1 to E125.

In a first embodiment of the present invention, biallelic markers are identified using genomic sequence information generated by the inventors. Sequenced genomic DNA fragments are used to design primers for the amplification of 500 bp fragments. These 500 bp fragments are amplified from genomic DNA and are scanned for biallelic markers. Primers may be designed using the OSP software (Hillier L. and Green P., 1991). All primers may contain, upstream of the specific target bases, a common oligonucleotide tail that serves as a sequencing primer. Those skilled in the art are familiar with primer extensions, which can be used for these purposes.

Preferred primers, useful for the amplification of genomic sequences encoding the candidate genes, focus on promoters, exons and splice sites of the genes. A biallelic marker presents a higher probability to be an eventual causal mutation if it is located in these functional regions of the gene. Preferred amplification primers of the invention include the nucleotide sequences B1 to B47 and C1 to C47, detailed further in Example 2, Table 1.

Sequencing of Amplified Genomic DNA and Identification of Single Nucleotide Polymorphisms

The amplification products generated as described above, are then sequenced using any method known and available to the skilled technician. Methods for sequencing DNA using either the dideoxy-mediated method (Sanger method) or the Maxam-Gilbert method are widely known to those of ordinary skill in the art. Such methods are for example disclosed in Sambrook et al.(1989). Alternative approaches include hybridization to high-density DNA probe arrays as described in Chee et al.(1996).

Preferably, the amplified DNA is subjected to automated dideoxy terminator sequencing reactions using a dye-primer cycle sequencing protocol. The products of the sequencing reactions are run on sequencing gels and the sequences are determined using gel image analysis. The polymorphism search is based on the presence of superimposed peaks in the electrophoresis pattern resulting from different bases occurring at the same position. Because each dideoxy terminator is labeled with a different fluorescent molecule, the two peaks corresponding to a biallelic site present distinct colors corresponding to two different nucleotides at the same position on the sequence. However, the presence of two peaks can be an artifact due to background noise. To exclude such an artifact, the two DNA strands are sequenced and a comparison between the peaks is carried out. In order to be registered as a polymorphic sequence, the polymorphism has to be detected on both strands.

The above procedure permits those amplification products, which contain biallelic markers to be identified. The detection limit for the frequency of biallelic polymorphisms detected by sequencing pools of 100 individuals is approximately 0.1 for the minor allele, as verified by sequencing pools of known allelic frequencies. However, more than 90% of the w biallelic polymorphisms detected by the pooling method have a frequency for the minor allele higher than 0.25. Therefore, the biallelic markers selected by this method have a frequency of at least 0.1 for the minor allele and less than 0.9 for the major allele. Preferably at least 0.2 for the minor allele and less than 0.8 for the major allele, more preferably at least 0.3 for the minor allele and less than 0.7 for the major allele, thus a heterozygosity rate higher than 0.18, preferably higher than 0.32, more preferably higher than 0.42.

In another embodiment, biallelic markers are detected by sequencing individual DNA samples, the frequency of the minor allele of such a biallelic marker may be less than 0. 1.

Validation of the Biallelic Markers of the Present Invention

The polymorphisms are evaluated for their usefulness as genetic markers by validating that both alleles are present in a population. Validation of the biallelic markers is accomplished by genotyping a group of individuals by a method of the invention and demonstrating that both alleles are present. Microsequencing is a preferred method of genotyping alleles. The validation by genotyping step may be performed on individual samples derived from each individual in the group or by genotyping a pooled sample derived from more than one individual. The group can be as small as one individual if that individual is heterozygous for the allele in question. Preferably the group contains at least three individuals, more preferably the group contains five or six individuals, so that a single validation test will be more likely to result in the validation of more of the biallelic markers that are being tested. It should be noted, however, that when the validation test is performed on a small group it may result in a false negative result if as a result of sampling error none of the individuals tested carries one of the two alleles. Thus, the validation process is less useful in demonstrating that a particular initial result is an artifact, than it is at demonstrating that there is a bonafide biallelic marker at a particular position in a sequence. All of the genotyping, haplotyping, association, and interaction study methods of the invention may optionally be performed solely with validated biallelic markers.

Evaluation of the Frequency of the Biallelic Markers of the Present Invention

The validated biallelic markers are further evaluated for their usefulness as genetic markers by determining the frequency of the least common allele at the biallelic marker site. The higher the frequency of the less common allele the greater the usefulness of the biallelic marker is association and interaction studies. The determination of the least common allele is accomplished by genotyping a group of individuals by a method of the invention and demonstrating that both alleles are present. This determination of frequency by genotyping step may be performed on individual samples derived from each individual in the group or by genotyping a pooled sample derived from more than one individual. The group must be large enough to be representative of the population as a whole. Preferably the group contains at least 20 individuals, more preferably the group contains at least 50 individuals, most preferably the group contains at least 100 individuals. Of course the larger the group the greater the accuracy of the frequency determination because of reduced sampling error. For an indication of the frequency for the less common allele of a particular biallelic marker of the invention see Table 2. A biallelic marker wherein the frequency of the less common allele is 30% or more is termed a “high quality biallelic marker.” All of the genotyping, haplotyping, association, and interaction study methods of the invention may optionally be performed solely with high quality biallelic markers.

Methods for Genotyping an Individual for Biallelic Markers

Methods are provided to genotype a biological sample for one or more biallelic markers of the present invention, all of which may be performed in vitro. Such methods of genotyping comprise determining the identity of a nucleotide at a PCTA-1 biallelic marker site by any method known in the art. These methods find use in genotyping case-control populations in association studies as well as individuals in the context of detection of alleles of biallelic markers which are known to be associated with a given trait, in which case both copies of the biallelic marker present in individual's genome are determined so that an individual may be classified as homozygous or heterozygous for a particular allele.

These genotyping methods can be performed on nucleic acid samples derived from a single individual or pooled DNA samples.

Genotyping can be performed using similar methods as those described above for the identification of the biallelic markers, or using other genotyping methods such as those further described below. In preferred embodiments, the comparison of sequences of amplified genomic fragments from different individuals is used to identify new biallelic markers whereas microsequencing is used for genotyping known biallelic markers in diagnostic and association study applications.

In one embodiment the invention encompasses methods of genotyping comprising determining the identity of a nucleotide at a PCTA-1-related biallelic marker or the complement thereof in a biological sample; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A44, A46 to A53, A57, A58, A62 to A76, A81, A82, A86 to A91, A107, A118, and A123 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A45, A54, A60, A61, A77 to A80, A83 to A85, A93, A102 to A106, A109, A122, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A55, A56, A59, A92, A94 to A01, A108, A111 to A113, A115 to A117, and A119 to A121, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said biological sample is derived from a single subject; optionally, wherein the identity of the nucleotides at said biallelic marker is determined for both copies of said biallelic marker present in said individual's genome; optionally, wherein said biological sample is derived from multiple subjects; Optionally, the genotyping methods of the invention encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination; Optionally, said method is performed in vitro; optionally, further comprising amplifying a portion of said sequence comprising the biallelic marker prior to said determining step; Optionally, wherein said amplifying is performed by PCR, LCR, or replication of a recombinant vector comprising an origin of replication and said fragment in a host cell; optionally, wherein said determining is performed by a hybridization assay, a sequencing assay, a microsequencing assay, or an enzyme-based mismatch detection assay.

Source of Nucleic Acids for Genotyping

Any source of nucleic acids, in purified or non-purified form, can be utilized as the starting nucleic acid, provided it contains or is suspected of containing the specific nucleic acid sequence desired. DNA or RNA may be extracted from cells, tissues, body fluids and the like as described above. While nucleic acids for use in the genotyping methods of the invention can be derived from any mammalian source, the test subjects and individuals from which nucleic acid samples are taken are generally understood to be human.

Amplification of DNA Fragments Comprising Biallelic Markers

Methods and polynucleotides are provided to amplify a segment of nucleotides comprising one or more biallelic marker of the present invention. It will be appreciated that amplification of DNA fragments comprising biallelic markers may be used in various methods and for various purposes and is not restricted to genotyping. Nevertheless, many genotyping methods, although not all, require the previous amplification of the DNA region carrying the biallelic marker of interest. Such methods specifically increase the concentration or total number of sequences that span the biallelic marker or include that site and sequences located either distal or proximal to it. Diagnostic assays may also rely on amplification of DNA segments carrying a biallelic marker of the present invention. Amplification of DNA may be achieved by any method known in the art. Amplification techniques are described above in the section entitled, “DNA amplification.”

The invention also concerns a method for the amplification of a PCTA-1 gene region, preferably containing at least one of the polymorphic bases identified in the context of the present invention, or a fragment or variant thereof, in a test sample. The method comprises the step of contacting a test sample suspected of containing the targeted PCTA-1 gene sequence or a fragment thereof with amplification reaction reagents comprising a pair of amplification primers, preferably located on either side of the polymorphic base. Preferred amplification primers consist of B1 to B47 and C1 to C47. The method may further comprise the step of detecting the amplification product. For example, the amplification product may be detected using a detection probe that can hybridize with an internal region of the amplicon sequences. In some embodiments, the polymorphic base is included in one of the sequences of P1 to P125, and the complementary sequences thereof.

Some of these amplification methods are particularly suited for the detection of single nucleotide polymorphisms and allow the simultaneous amplification of a target sequence and the identification of the polymorphic nucleotide as it is further described below.

The identification of biallelic markers as described above allows the design of appropriate oligonucleotides, which can be used as primers to amplify DNA fragments comprising the biallelic markers of the present invention. Amplification can be performed using the primers initially used to discover new biallelic markers which are described herein or any set of primers allowing the amplification of a DNA fragment comprising a biallelic marker of the present invention.

In some embodiments the present invention provides primers for amplifying a DNA fragment containing one or more biallelic markers of the present invention. Preferred amplification primers are listed in Example 2. It will be appreciated that the primers listed are merely exemplary and that any other set of primers which produce amplification products containing one or more biallelic markers of the present invention are also of use.

The spacing of the primers determines the length of the segment to be amplified. In the context of the present invention, amplified segments carrying biallelic markers can range in size from at least about 25 bp to 35 kbp. Amplification fragments from 25-3000 bp are typical, fragments from 50-1000 bp are preferred and fragments from 100-600 bp are highly preferred. It will be appreciated that amplification primers for the biallelic markers may be any sequence which allow the specific amplification of any DNA fragment carrying the markers. Amplification primers may be labeled or immobilized on a solid support as described in “Oligonucleotide probes and primers”.

Methods of Genotyping DNA Samples for Biallelic Markers

Any method known in the art can be used to identify the nucleotide present at a biallelic marker site. Since the biallelic marker allele to be detected has been identified and specified in the present invention, detection will prove simple for one of ordinary skill in the art by employing any of a number of techniques. Many genotyping methods require the previous amplification of the DNA region carrying the biallelic marker of interest. While the amplification of target or signal is often preferred at present, ultrasensitive detection methods which do not require amplification are also encompassed by the present genotyping methods. Methods well-known to those skilled in the art that can be used to detect biallelic polymorphisms include methods such as, conventional dot blot analyzes, single strand conformational polymorphism analysis (SSCP) described by Orita et al.(1989), denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis, mismatch cleavage detection, and other conventional techniques as described in Sheffield et al.(1991), White et al.(1992), Grompe et al.(1989 and 1993). Another method for determining the identity of the nucleotide present at a particular polymorphic site employs a specialized exonuclease-resistant nucleotide derivative as described in U.S. Pat. No. 4,656,127.

Preferred methods involve directly determining the identity of the nucleotide present at a biallelic marker site by sequencing assay, enzyme-based mismatch detection assay, or hybridization assay. The following is a description of some preferred methods. A highly preferred method is the microsequencing technique. The term “sequencing” is generally used herein to refer to polymerase extension of duplex primer/template complexes and includes both traditional sequencing and microsequencing.

1) Sequencing Assays

The nucleotide present at a polymorphic site can be determined by sequencing methods. In a preferred embodiment, DNA samples are subjected to PCR amplification before sequencing as described above. DNA sequencing methods are described in “Sequencing Of Amplified Genomic DNA And Identification Of Single Nucleotide Polymorphisms”.

Preferably, the amplified DNA is subjected to automated dideoxy terminator sequencing reactions using a dye-primer cycle sequencing protocol. Sequence analysis allows the identification of the base present at the biallelic marker site.

2) Microsequencing Assays

In microsequencing methods, the nucleotide at a polymorphic site in a target DNA is detected by a single nucleotide primer extension reaction. This method involves appropriate microsequencing primers which, hybridize just upstream of the polymorphic base of interest in the target nucleic acid. A polymerase is used to specifically extend the 3′ end of the primer with one single ddNTP (chain terminator) complementary to the nucleotide at the polymorphic site. Next the identity of the incorporated nucleotide is determined in any suitable way.

Typically, microsequencing reactions are carried out using fluorescent ddNTPs and the extended microsequencing primers are analyzed by electrophoresis on ABI 377 sequencing machines to determine the identity of the incorporated nucleotide as described in EP412 883, the disclosure of which is incorporated herein by reference in its entirety. Alternatively capillary electrophoresis can be used in order to process a higher number of assays simultaneously. An example of a typical microsequencing procedure that can be used in the context of the present invention is provided in Example 4.

Different approaches can be used for the labeling and detection of ddNTPs. A homogeneous phase detection method based on fluorescence resonance energy transfer has been described by Chen and Kwok (1997) and Chen et al.(1997). In this method, amplified genomic DNA fragments containing polymorphic sites are incubated with a 5′-fluorescein-labeled primer in the presence of allelic dye-labeled dideoxyribonucleoside triphosphates and a modified Taq polymerase. The dye-labeled primer is extended one base by the dye-terminator specific for the allele present on the template. At the end of the genotyping reaction, the fluorescence intensities of the two dyes in the reaction mixture are analyzed directly without separation or purification. All these steps can be performed in the same tube and the fluorescence changes can be monitored in real time. Alternatively, the extended primer may be analyzed by MALDI-TOF Mass Spectrometry. The base at the polymorphic site is identified by the mass added onto the microsequencing primer (see Haff and Smirnov, 1997).

Microsequencing may be achieved by the established microsequencing method or by developments or derivatives thereof. Alternative methods include several solid-phase microsequencing techniques. The basic microsequencing protocol is the same as described previously, except that the method is conducted as a heterogeneous phase assay, in which the primer or the target molecule is immobilized or captured onto a solid support. To simplify the primer separation and the terminal nucleotide addition analysis, oligonucleotides are attached to solid supports or are modified in such ways that permit affinity separation as well as polymerase extension. The 5′ ends and internal nucleotides of synthetic oligonucleotides can be modified in a number of different ways to permit different affinity separation approaches, e.g., biotinylation. If a single affinity group is used on the oligonucleotides, the oligonucleotides can be separated from the incorporated terminator regent. This eliminates the need of physical or size separation. More than one oligonucleotide can be separated from the terminator reagent and analyzed simultaneously if more than one affinity group is used. This permits the analysis of several nucleic acid species or more nucleic acid sequence information per extension reaction. The affinity group need not be on the priming oligonucleotide but could alternatively be present on the template. For example, immobilization can be carried out via an interaction between biotinylated DNA and streptavidin-coated microtitration wells or avidin-coated polystyrene particles. In the same manner, oligonucleotides or templates may be attached to a solid support in a high-density format. In such solid phase microsequencing reactions, incorporated ddNTPs can be radiolabeled (Syvänen, 1994) or linked to fluorescein (Livak and Hainer, 1994). The detection of radiolabeled ddNTPs can be achieved through scintillation-based techniques. The detection of fluorescein-linked ddNTPs can be based on the binding of antifluorescein antibody conjugated with alkaline phosphatase, followed by incubation with a chromogenic substrate (such asp-nitrophenyl phosphate). Other possible reporter-detection pairs include: ddNTP linked to dinitrophenyl (DNP) and anti-DNP alkaline phosphatase conjugate (Harju et al., 1993) or biotinylated ddNTP and horseradish peroxidase-conjugated streptavidin with o-phenylenediamine as a substrate (WO 92/15712, the disclosure of which is incorporated herein by reference in its entirety). As yet another alternative solid-phase microsequencing procedure, Nyren et al.(1993) described a method relying on the detection of DNA polymerase activity by an enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA).

Pastinen et al.(1997) describe a method for multiplex detection of single nucleotide polymorphism in which the solid phase minisequencing principle is applied to an oligonucleotide array format. High-density arrays of DNA probes attached to a solid support (DNA chips) are further described below.

In one aspect the present invention provides polynucleotides and methods to genotype one or more biallelic markers of the present invention by performing a microsequencing assay. Preferred microsequencing primers include the nucleotide sequences D1 to D125 and E1 to E125. More preferred microsequencing primers are selected from the group consisting of the nucleotide sequences D15, D24, D30, D34, D36, D38, D41, D44, D50, D53, D54, D56, D57, D59, D76, D85, D93, D108, D111, D115, D124, Eli, E14, E22, E25, E26, E35, E42, E52, E53, E55, E56, E60, E61, E64, E73, E75, E93, E96. It will be appreciated that the microsequencing primers listed in Example 4 are merely exemplary and that, any primer having a 3′ end immediately adjacent to the polymorphic nucleotide may be used. Similarly, it will be appreciated that microsequencing analysis may be performed for any biallelic marker or any combination of biallelic markers of the present invention. One aspect of the present invention is a solid support which includes one or more microsequencing primers listed in Example 4, or fragments comprising at least 8, 12, 15, 20, 25, 30, 40, or 50 consecutive nucleotides thereof, to the extent that such lengths are consistent with the primer described, and having a 3′ terminus immediately upstream of the corresponding biallelic marker, for determining the identity of a nucleotide at a biallelic marker site.

3) Mismatch Detection Assays Based on Polymerases and Ligases

In one aspect the present invention provides polynucleotides and methods to determine the allele of one or more biallelic markers of the present invention in a biological sample, by mismatch detection assays based on polymerases and/or ligases. These assays are based on the specificity of polymerases and ligases. Polymerization reactions places particularly stringent requirements on correct base pairing of the 3′ end of the amplification primer and the joining of two oligonucleotides hybridized to a target DNA sequence is quite sensitive to mismatches close to the ligation site, especially at the 3′ end. Methods, primers and various parameters to amplify DNA fragments comprising biallelic markers of the present invention are further described above in “Amplification Of DNA Fragments Comprising Biallelic Markers”.

Allele Specific Amplification Primers

Discrimination between the two alleles of a biallelic marker can also be achieved by allele specific amplification, a selective strategy, whereby one of the alleles is amplified without amplification of the other allele. For allele specific amplification, at least one member of the pair of primers is sufficiently complementary with a region of a PCTA-1 gene comprising the polymorphic base of a biallelic marker of the present invention to hybridize therewith and to initiate the amplification. Such primers are able to discriminate between the two alleles of a biallelic marker.

This is accomplished by placing the polymorphic base at the 3′ end of one of the amplification primers. Because the extension forms from the 3′ end of the primer, a mismatch at or near this position has an inhibitory effect on amplification. Therefore, under appropriate amplification conditions, these primers only direct amplification on their complementary allele. Determining the precise location of the mismatch and the corresponding assay conditions are well within the ordinary skill in the art.

Ligation/Amplification Based Methods

The “Oligonucleotide Ligation Assay” (OLA) uses two oligonucleotides which are designed to be capable of hybridizing to abutting sequences of a single strand of a target molecules. One of the oligonucleotides is biotinylated, and the other is detectably labeled. If the precise complementary sequence is found in a target molecule, the oligonucleotides will hybridize such that their termini abut, and create a ligation substrate that can be captured and detected. OLA is capable of detecting single nucleotide polymorphisms and may be advantageously combined with PCR as described by Nickerson et al.(1990). In this method, PCR is used to achieve the exponential amplification of target DNA, which is then detected using OLA.

Other amplification methods which are particularly suited for the detection of single nucleotide polymorphism include LCR (ligase chain reaction), Gap LCR (GLCR) which are described above in “DNA Amplification”. LCR uses two pairs of probes to exponentially amplify a specific target. The sequences of each pair of oligonucleotides, is selected to permit the pair to hybridize to abutting sequences of the same strand of the target. Such hybridization forms a substrate for a template-dependant ligase. In accordance with the present invention, LCR can be performed with oligonucleotides having the proximal and distal sequences of the same strand of a biallelic marker site. In one embodiment, either oligonucleotide will be designed to include the biallelic marker site. In such an embodiment, the reaction conditions are selected such that the oligonucleotides can be ligated together only if the target molecule either contains or lacks the specific nucleotide that is complementary to the biallelic marker on the oligonucleotide. In an alternative embodiment, the oligonucleotides will not include the biallelic marker, such that when they hybridize to the target molecule, a “gap” is created as described in WO 90/01069, the disclosure of which is incorporated herein by reference in its entirety. This gap is then “filled” with complementary dNTPs (as mediated by DNA polymerase), or by an additional pair of oligonucleotides. Thus at the end of each cycle, each single strand has a complement capable of serving as a target during the next cycle and exponential allele-specific amplification of the desired sequence is obtained.

Ligase/Polymerase-mediated Genetic Bit Analysis™ is another method for determining the identity of a nucleotide at a preselected site in a nucleic acid molecule (WO 95/21271, the disclosure of which is incorporated herein by reference in its entirety). This method involves the incorporation of a nucleoside triphosphate that is complementary to the nucleotide present at the preselected site onto the terminus of a primer molecule, and their subsequent ligation to a second oligonucleotide. The reaction is monitored by detecting a specific label attached to the reaction's solid phase or by detection in solution.

4) Hybridization Assay Methods

A preferred method of determining the identity of the nucleotide present at a biallelic marker site involves nucleic acid hybridization. The hybridization probes, which can be conveniently used in such reactions, preferably include the probes defined herein. Any hybridization assay may be used including Southern hybridization, Northern hybridization, dot blot hybridization and solid-phase hybridization (see Sambrook et al., 1989).

Hybridization refers to the formation of a duplex structure by two single stranded nucleic acids due to complementary base pairing. Hybridization can occur between exactly complementary nucleic acid strands or between nucleic acid strands that contain minor regions of mismatch. Specific probes can be designed that hybridize to one form of a biallelic marker and not to the other and therefore are able to discriminate between different allelic forms. Allele-specific probes are often used in pairs, one member of a pair showing perfect match to a target sequence containing the original allele and the other showing a perfect match to the target sequence containing the alternative allele. Hybridization conditions should be sufficiently stringent that there is a significant difference in hybridization intensity between alleles, and preferably an essentially binary response, whereby a probe hybridizes to only one of the alleles. Stringent, sequence specific hybridization conditions, under which a probe will hybridize only to the exactly complementary target sequence are well known in the art (Sambrook et al., 1989). Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Although such hybridization can be performed in solution, it is preferred to employ a solid-phase hybridization assay. The target DNA comprising a biallelic marker of the present invention may be amplified prior to the hybridization reaction. The presence of a specific allele in the sample is determined by detecting the presence or the absence of stable hybrid duplexes formed between the probe and the target DNA. The detection of hybrid duplexes can be carried out by a number of methods. Various detection assay formats are well known which utilize detectable labels bound to either the target or the probe to enable detection of the hybrid duplexes. Typically, hybridization duplexes are separated from unhybridized nucleic acids and the labels bound to the duplexes are then detected. Those skilled in the art will recognize that wash steps may be employed to wash away excess target DNA or probe as well as unbound conjugate. Further, standard heterogeneous assay formats are suitable for detecting the hybrids using the labels present on the primers and probes.

Two recently developed assays allow hybridization-based allele discrimination with no need for separations or washes (see Landegren U. et al., 1998). The TaqMan assay takes advantage of the 5′ nuclease activity of Taq DNA polymerase to digest a DNA probe annealed specifically to the accumulating amplification product. TaqMan probes are labeled with a donor-acceptor dye pair that interacts via fluorescence energy transfer. Cleavage of the TaqMan probe by the advancing polymerase during amplification dissociates the donor dye from the quenching acceptor dye, greatly increasing the donor fluorescence. All reagents necessary to detect two allelic variants can be assembled at the beginning of the reaction and the results are monitored in real time (see Livak et al., 1995). In an alternative homogeneous hybridization based procedure, molecular beacons are used for allele discriminations. Molecular beacons are hairpin-shaped oligonucleotide probes that report the presence of specific nucleic acids in homogeneous solutions. When they bind to their targets they undergo a conformational reorganization that restores the fluorescence of an internally quenched fluorophore (Tyagi et al., 1998).

The polynucleotides provided herein can be used to produce probes which can be used in hybridization assays for the detection of biallelic marker alleles in biological samples. These probes are characterized in that they preferably comprise between 8 and 50 nucleotides, and in that they are sufficiently complementary to a sequence comprising a biallelic marker of the present invention to hybridize thereto and preferably sufficiently specific to be able to discriminate the targeted sequence for only one nucleotide variation. A particularly preferred probe is 25 nucleotides in length. Another particularly preferred probe is 47 nucleotides in length. Preferably the biallelic marker is within 4 nucleotides of the center of the polynucleotide probe. In particularly preferred probes, the biallelic marker is at the center of said polynucleotide. Preferred probes comprise a nucleotide sequence selected from the group consisting of amplicons listed in Table 1 and the sequences complementary thereto, or a fragment thereof, said fragment comprising at least about 8 consecutive nucleotides, preferably 10, 15, 20, more preferably 25, 30, 40, 47, or 50 consecutive nucleotides and containing a polymorphic base. Preferred probes comprise a nucleotide sequence selected from the group consisting of P1 to P125 and the sequences complementary thereto. In preferred embodiments the polymorphic base(s) are within 5, 4, 3, 2, 1, nucleotides of the center of the said polynucleotide, more preferably at the center of said polynucleotide.

Preferably the probes of the present invention are labeled or immobilized on a solid support. Labels and solid supports are further described in “Oligonucleotide Probes and Primers”. The probes can be non-extendable as described in “Oligonucleotide Probes and Primers”.

By assaying the hybridization to an allele specific probe, one can detect the presence or absence of a biallelic marker allele in a given sample. High-Throughput parallel hybridization in array format is specifically encompassed within “hybridization assays” and are described below.

5) Hybridization to Addressable Arrays of Oligonucleotides

Hybridization assays based on oligonucleotide arrays rely on the differences in hybridization stability of short oligonucleotides to perfectly matched and mismatched target sequence variants. Efficient access to polymorphism information is obtained through a basic structure comprising high-density arrays of oligonucleotide probes attached to a solid support (e.g., the chip) at selected positions. Each DNA chip can contain thousands to millions of individual synthetic DNA probes arranged in a grid-like pattern and miniaturized to the size of a dime.

The chip technology has already been applied with success in numerous cases. For example, the screening of mutations has been undertaken in the BRCA1 gene, in S. cerevisiae mutant strains, and in the protease gene of HIV-1 virus (Hacia et al., 1996; Shoemaker et al., 1996; Kozal et al., 1996). Chips of various formats for use in detecting biallelic polymorphisms can be produced on a customized basis by Affymetrix (GeneChip™), Hyseq (HyChip and HyGnostics), and Protogene Laboratories.

In general, these methods employ arrays of oligonucleotide probes that are complementary to target nucleic acid sequence segments from an individual which, target sequences include a polymorphic marker. EP785280, the disclosure of which is incorporated herein by reference in its entirety, describes a tiling strategy for the detection of single nucleotide polymorphisms. Briefly, arrays may generally be “tiled” for a large number of specific polymorphisms. By “tiling” is generally meant the synthesis of a defined set of oligonucleotide probes which is made up of a sequence complementary to the target sequence of interest, as well as preselected variations of that sequence, e.g., substitution of one or more given positions with one or more members of the basis set of nucleotides. Tiling strategies are further described in PCT application No. WO 95/11995, the disclosure of which is incorporated herein by reference in its entirety. In a particular aspect, arrays are tiled for a number of specific, identified biallelic marker sequences. In particular, the array is tiled to include a number of detection blocks, each detection block being specific for a specific biallelic marker or a set of biallelic markers. For example, a detection block may be tiled to include a number of probes, which span the sequence segment that includes a specific polymorphism. To ensure probes that are complementary to each allele, the probes are synthesized in pairs differing at the biallelic marker. In addition to the probes differing at the polymorphic base, monosubstituted probes are also generally tiled within the detection block. These monosubstituted probes have bases at and up to a certain number of bases in either direction from the polymorphism, substituted with the remaining nucleotides (selected from A, T, G, C and U). Typically the probes in a tiled detection block will include substitutions of the sequence positions up to and including those that are 5 bases away from the biallelic marker. The monosubstituted probes provide internal controls for the tiled array, to distinguish actual hybridization from artefactual cross-hybridization. Upon completion of hybridization with the target sequence and washing of the array, the array is scanned to determine the position on the array to which the target sequence hybridizes. The hybridization data from the scanned array is then analyzed to identify which allele or alleles of the biallelic marker are present in the sample. Hybridization and scanning may be carried out as described in PCT application No. WO 92/10092 and WO 95/11995 and U.S. Pat. No. 5,424,186, the disclosures of which are incorporated herein by reference in their entireties.

Thus, in some embodiments, the chips may comprise an array of nucleic acid sequences of fragments of about IS nucleotides in length. In further embodiments, the chip may comprise an array including at least one of the sequences selected from the group consisting of amplicons listed in table I and the sequences complementary thereto, or a fragment thereof, said fragment comprising at least about 8 consecutive nucleotides, preferably 10, 15, 20, more preferably 25, 30, 40, 47, or 50 consecutive nucleotides and containing a polymorphic base. In preferred embodiments the polymorphic base is within 5, 4, 3, 2, 1, nucleotides of the center of the said polynucleotide, more preferably at the center of said polynucleotide. In some embodiments, the chip may comprise an array of at least 2, 3, 4, 5, 6, 7, 8 or more of these polynucleotides of the invention. Solid supports and polynucleotides of the present invention attached to solid supports are further described in “Oligonucleotide Probes And Primers”.

6) Integrated Systems

Another technique, which may be used to analyze polymorphisms, includes multicomponent integrated systems, which miniaturize and compartmentalize processes such as PCR and capillary electrophoresis reactions in a single functional device. An example of such technique is disclosed in US patent 5,589,136, the disclosure of which is incorporated herein by reference in its entirety, which describes the integration of PCR amplification and capillary electrophoresis in chips.

Integrated systems can be envisaged mainly when microfluidic systems are used. These systems comprise a pattern of microchannels designed onto a glass, silicon, quartz, or plastic wafer included on a microchip. The movements of the samples are controlled by electric, electroosmotic or hydrostatic forces applied across different areas of the microchip to create functional microscopic valves and pumps with no moving parts.

For genotyping biallelic markers, the microfluidic system may integrate nucleic acid amplification, microsequencing, capillary electrophoresis and a detection method such as laser-induced fluorescence detection.

Methods of Genetic Analysis using the Biallelic Markers of the Present Invention

Different methods are available for the genetic analysis of complex traits (see Lander and Schork, 1994). The search for disease-susceptibility genes is conducted using two main methods: the linkage approach in which evidence is sought for cosegregation between a locus and a putative trait locus using family studies, and the association approach in which evidence is sought for a statistically significant association between an allele or a trait causing allele and a trait (Khoury et al., 1993). In general, the biallelic markers of the present invention find use in any method known in the art to demonstrate a statistically significant correlation between a genotype and a phenotype. The biallelic markers may be used in parametric and non-parametric linkage analysis methods. Preferably, the biallelic markers of the present invention are used to identify genes associated with detectable traits using association studies, an approach which does not require the use of affected families and which permits the identification of genes associated with complex and sporadic traits.

The genetic analysis using the biallelic markers of the present invention may be conducted on any scale. The whole set of biallelic markers of the present invention or any subset of biallelic markers of the present invention corresponding to the candidate gene may be used. Further, any set of genetic markers including a biallelic marker of the present invention may be used. A set of biallelic polymorphisms that could be used as genetic markers in combination with the biallelic markers of the present invention has been described in WO 98120165, the disclosure of which is incorporated herein by reference in its entirety. As mentioned above, it should be noted that the biallelic markers of the present invention may be included in any complete or partial genetic map of the human genome. These different uses are specifically contemplated in the present invention and claims.

Linkage Analysis

Linkage analysis is based upon establishing a correlation between the transmission of genetic markers and that of a specific trait throughout generations within a family. Thus, the aim of linkage analysis is to detect marker loci that show cosegregation with a trait of interest in pedigrees.

Parametric Methods

When data are available from successive generations there is the opportunity to study the degree of linkage between pairs of loci. Estimates of the recombination fraction enable loci to be ordered and placed onto a genetic map. With loci that are genetic markers, a genetic map can be established, and then the strength of linkage between markers and traits can be calculated and used to indicate the relative positions of markers and genes affecting those traits (Weir, 1996). The classical method for linkage analysis is the logarithm of odds (lod) score method (see Morton, 1955; Ott, 1991). Calculation of lod scores requires specification of the mode of inheritance for the disease (parametric method). Generally, the length of the candidate region identified using linkage analysis is between 2 and 20 Mb. Once a candidate region is identified as described above, analysis of recombinant individuals using additional markers allows further delineation of the candidate region. Linkage analysis studies have generally relied on the use of a maximum of 5,000 microsatellite markers, thus limiting the maximum theoretical attainable resolution of linkage analysis to about 600 kb on average.

Linkage analysis has been successfully applied to map simple genetic traits that show clear Mendelian inheritance patterns and which have a high penetrance (i.e., the ratio between the number of trait positive carriers of allele a and the total number of a carriers in the population). However, parametric linkage analysis suffers from a variety of drawbacks. First, it is limited by its reliance on the choice of a genetic model suitable for each studied trait. Furthermore, as already mentioned, the resolution attainable using linkage analysis is limited, and complementary studies are required to refine the analysis of the typical 2 Mb to 20 Mb regions initially identified through linkage analysis. In addition, parametric linkage analysis approaches have proven difficult when applied to complex genetic traits, such as those due to the combined action of multiple genes and/or environmental factors. It is very difficult to model these factors adequately in a lod score analysis. In such cases, too large an effort and cost are needed to recruit the adequate number of affected families required for applying linkage analysis to these situations, as recently discussed by Risch, N. and Merikangas, K. (1 996).

Non-Parametric Methods

The advantage of the so-called non-parametric methods for linkage analysis is that they do not require specification of the mode of inheritance for the disease, they tend to be more useful for the analysis of complex traits. In non-parametric methods, one tries to prove that the inheritance pattern of a chromosomal region is not consistent with random Mendelian segregation by showing that affected relatives inherit identical copies of the region more often than expected by chance. Affected relatives should show excess “allele sharing” even in the presence of incomplete penetrance and polygenic inheritance. In non-parametric linkage analysis the degree of agreement at a marker locus in two individuals can be measured either by the number of alleles identical by state (IBS) or by the number of alleles identical by descent (IBD). Affected sib pair analysis is a well-known special case and is the simplest form of these methods.

The biallelic markers of the present invention may be used in both parametric and non-parametric linkage analysis. Preferably biallelic markers may be used in non-parametric methods which allow the mapping of genes involved in complex traits. The biallelic markers of the present invention may be used in both IBD- and IBS-methods to map genes affecting a complex trait. In such studies, taking advantage of the high density of biallelic markers, several adjacent biallelic marker loci may be pooled to achieve the efficiency attained by multi-allelic markers (Zhao et al., 1998).

Population Association Studies

The present invention comprises methods for identifying if the PCTA-1 gene is associated with a detectable trait using the biallelic markers of the present invention. In one embodiment the present invention comprises methods to detect an association between a biallelic marker allele or a biallelic marker haplotype and a trait. Further, the invention comprises methods to identify a trait causing allele in linkage disequilibrium with any biallelic marker allele of the present invention.

Alternative approaches can be employed to perform association studies: genome-wide association studies, candidate region association studies and candidate gene association studies. In a preferred embodiment, the biallelic markers of the present invention are used to perform candidate gene association studies. The candidate gene analysis clearly provides a short-cut approach to the identification of genes and gene polymorphisms related to a particular trait when some information concerning the biology of the trait is available. Further, the biallelic markers of the present invention may be incorporated in any map of genetic markers of the human genome in order to perform genome-wide association studies. Methods to generate a high-density map of biallelic markers has been described in U.S. Provisional Patent application serial No. 60/082,614. The biallelic markers of the present invention may further be incorporated in any map of a specific candidate region of the genome (a specific chromosome or a specific chromosomal segment for example).

As mentioned above, association studies may be conducted within the general population and are not limited to studies performed on related individuals in affected families. Association studies are extremely valuable as they permit the analysis of sporadic or multifactor traits. Moreover, association studies represent a powerful method for fine-scale mapping enabling much finer mapping of trait causing alleles than linkage studies. Studies based on pedigrees often only narrow the location of the trait causing allele. Association studies using the biallelic markers of the present invention can therefore be used to refine the location of a trait causing allele in a candidate region identified by Linkage Analysis methods. Moreover, once a chromosome segment of interest has been identified, the presence of a candidate gene such as a candidate gene of the present invention, in the region of interest can provide a shortcut to the identification of the trait causing allele. Biallelic markers of the present invention can be used to demonstrate that a candidate gene is associated with a trait. Such uses are specifically contemplated in the present invention.

Determining the Frequency of a Biallelic Marker Allele or of a Biallelic Marker Haplotype in a Population

Association studies explore the relationships among frequencies for sets of alleles between loci.

Determining the Frequency of an Allele in a Population

Allelic frequencies of the biallelic markers in a populations can be determined using one of the methods described above under the heading “Methods For Genotyping An Individual For Biallelic Markers”, or any genotyping procedure suitable for this intended purpose. Genotyping pooled samples or individual samples can determine the frequency of a biallelic marker allele in a population. One way to reduce the number of genotypings required is to use pooled samples. A major obstacle in using pooled samples is in terms of accuracy and reproducibility for determining accurate DNA concentrations in setting up the pools. Genotyping individual samples provides higher sensitivity, reproducibility and accuracy and; is the preferred method used in the present invention. Preferably, each individual is genotyped separately and simple gene counting is applied to determine the frequency of an allele of a biallelic marker or of a genotype in a given population.

The invention also relates to methods of estimating the frequency of a PCTA-1-related biallelic marker allele in a population comprising: a) genotyping individuals from said population for said biallelic marker according to the method of the present invention; and b) determining the proportional representation of said biallelic marker in said population. In addition, the methods of estimating the frequency of an allele in a population of the invention encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A44, A46 to A53, A57, A58, A62 to A76, A81, A82, A86 to A91, A107, A118, and A123 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A45, A54, A60, A61, A77 to A80, A83 to A85, A93, A102 to A106, A109, A110, A114, and A122, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A55, A56, A59, A92, A94 to A101, A108, A111 to A113, A115 to A117, and A119 to A121, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; Optionally, determining the frequency of a biallelic marker allele in a population may be accomplished by determining the identity of the nucleotides for both copies of said biallelic marker present in the genome of each individual in said population and calculating the proportional representation of said nucleotide at said PCTA-1-related biallelic marker for the population; Optionally, determining the proportional representation may be accomplished by performing a genotyping method of the invention on a pooled biological sample derived from a representative number of individuals, or each individual, in said population, and calculating the proportional amount of said nucleotide compared with the total.

Determining the Frequency of a Haplotype in a Population

The gametic phase of haplotypes is unknown when diploid individuals are heterozygous at more than one locus. Using genealogical information in families gametic phase can sometimes be inferred (Perlin et al., 1994). When no genealogical information is available different strategies may be used. One possibility is that the multiple-site heterozygous diploids can be eliminated from the analysis, keeping only the homozygotes and the single-site heterozygote individuals, but this approach might lead to a possible bias in the sample composition and the underestimation of low-frequency haplotypes. Another possibility is that single chromosomes can be studied independently, for example, by asymmetric PCR amplification (see Newton et al, 1989; Wu et al., 1989) or by isolation of single chromosome by limit dilution followed by PCR amplification (see Ruano et al., 1990). Further, a sample may be haplotyped for sufficiently close biallelic markers by double PCR amplification of specific alleles (Sarkar, G. and Sommer S. S., 1991). These approaches are not entirely satisfying either because of their technical complexity, the additional cost they entail, their lack of generalization at a large scale, or the possible biases they introduce. To overcome these difficulties, an algorithm to infer the phase of PCR-amplified DNA genotypes introduced by Clark, A. G.(1990) may be used. Briefly, the principle is to start filling a preliminary list of haplotypes present in the sample by examining unambiguous individuals, that is, the complete homozygotes and the single-site heterozygotes. Then other individuals in the same sample are screened for the possible occurrence of previously recognized haplotypes. For each positive identification, the complementary haplotype is added to the list of recognized haplotypes, until the phase information for all individuals is either resolved or identified as unresolved. This method assigns a single haplotype to each multiheterozygous individual, whereas several haplotypes are possible when there are more than one heterozygous site. Alternatively, one can use methods estimating haplotype frequencies in a population without assigning haplotypes to each individual. Preferably, a method based on an expectation-maximization (EM) algorithm (Dempster et al., 1977) leading to maximum-likelihood estimates of haplotype frequencies under the assumption of Hardy-Weinberg proportions (random mating) is used (see Excoffier L. and Slatkin M., 1995). The EM algorithm is a generalized iterative maximum-likelihood approach to estimation that is useful when data are ambiguous and/or incomplete. The EM algorithm is used to resolve heterozygotes into haplotypes. Haplotype estimations are further described below under the heading “Statistical Methods.” Any other method known in the art to determine or to estimate the frequency of a haplotype in a population may be used.

The invention also encompasses methods of estimating the frequency of a haplotype for a set of biallelic markers in a population, comprising the steps of: a) genotyping at least one PCTA-1-related biallelic marker according to a method of the invention for each individual in said population; b) genotyping a second biallelic marker by determining the identity of the nucleotides at said second biallelic marker for both copies of said second biallelic marker present in the genome of each individual in said population; and c) applying a haplotype determination method to the identities of the nucleotides determined in steps a) and b) to obtain an estimate of said frequency. In addition, the methods of estimating the frequency of a haplotype of the invention encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A44, A46 to A53, A57, A58, A62 to A76, A81, A82, A86 to A91, A107, A118, and A123 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A45, A54, A60, A61, A77 to A80, A83 to A85, A93, A102 to A106, A109, A110, A114, and A122, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A55, A56, A59, A92, A94 to A101, A108, A111 to A113, A115 to A117, and A119 to A121, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; Optionally, said haplotype determination method is performed by asymmetric PCR amplification, double PCR amplification of specific alleles, the Clark algorithm, or an expectation-maximization algorithm.

Linkage Disequilibrium Analysis

Linkage disequilibrium is the non-random association of alleles at two or more loci and represents a powerful tool for mapping genes involved in disease traits (see Ajioka R. S. et al., 1997). Biallelic markers, because they are densely spaced in the human genome and can be genotyped in greater numbers than other types of genetic markers (such as RFLP or VNTR markers), are particularly useful in genetic analysis based on linkage disequilibrium.

When a disease mutation is first introduced into a population (by a new mutation or the immigration of a mutation carrier), it necessarily resides on a single chromosome and thus on a single “background” or “ancestral” haplotype of linked markers. Consequently, there is complete disequilibrium between these markers and the disease mutation: one finds the disease mutation only in the presence of a specific set of marker alleles. Through subsequent generations recombination events occur between the disease mutation and these marker polymorphisms, and the disequilibrium gradually dissipates. The pace of this dissipation is a function of the recombination frequency, so the markers closest to the disease gene will manifest higher levels of disequilibrium than those that are further away. When not broken up by recombination, “ancestral” haplotypes and linkage disequilibrium between marker alleles at different loci can be tracked not only through pedigrees but also through populations. Linkage disequilibrium is usually seen as an association between one specific allele at one locus and another specific allele at a second locus.

The pattern or curve of disequilibrium between disease and marker loci is expected to exhibit a maximum that occurs at the disease locus. Consequently, the amount of linkage disequilibrium between a disease allele and closely linked genetic markers may yield valuable information regarding the location of the disease gene. For fine-scale mapping of a disease locus, it is useful to have some knowledge of the patterns of linkage disequilibrium that exist between markers in the studied region. As mentioned above the mapping resolution achieved through the analysis of linkage disequilibrium is much higher than that of linkage studies. The high density of biallelic markers combined with linkage disequilibrium analysis provides powerful tools for fine-scale mapping. Different methods to calculate linkage disequilibrium are described below under the heading “Statistical Methods”.

Population-Based Case-Control Studies of Trait-Marker Associations

As mentioned above, the occurrence of pairs of specific alleles at different loci on the same chromosome is not random and the deviation from random is called linkage disequilibrium. Association studies focus on population frequencies and rely on the phenomenon of linkage disequilibrium. If a specific allele in a given gene is directly involved in causing a particular trait, its frequency will be statistically increased in an affected (trait positive) population, when compared to the frequency in a trait negative population or in a random control population. As a consequence of the existence of linkage disequilibrium, the frequency of all other alleles present in the haplotype carrying the trait-causing allele will also be increased in trait positive individuals compared to trait negative individuals or random controls. Therefore, association between the trait and any allele (specifically a biallelic marker allele) in linkage disequilibrium with the trait-causing allele will suffice to suggest the presence of a trait-related gene in that particular region. Case-control populations can be genotyped for biallelic markers to identify associations that narrowly locate a trait causing allele. As any marker in linkage disequilibrium with one given marker associated with a trait will be associated with the trait. Linkage disequilibrium allows the relative frequencies in case-control populations of a limited number of genetic polymorphisms (specifically biallelic markers) to be analyzed as an alternative to screening all possible functional polymorphisms in order to find trait-causing alleles. Association studies compare the frequency of marker alleles in unrelated case-control populations, and represent powerful tools for the dissection of complex traits.

Case-Control Populations (Inclusion Criteria)

Population-based association studies do not concern familial inheritance but compare the prevalence of a particular genetic marker, or a set of markers, in case-control populations. They are case-control studies based on comparison of unrelated case (affected or trait positive) individuals and unrelated control (unaffected, trait negative or random) individuals. Preferably the control group is composed of unaffected or trait negative individuals. Further, the control group is ethnically matched to the case population. Moreover, the control group is preferably matched to the case-population for the main known confusion factor for the trait under study (for example age-matched for an age-dependent trait). Ideally, individuals in the two samples are paired in such a way that they are expected to differ only in their disease status. The terms “trait positive population”, “case population” and “affected population” are used interchangeably herein.

An important step in the dissection of complex traits using association studies is the choice of case-control populations (see Lander and Schork, 1994). A major step in the choice of case-control populations is the clinical definition of a given trait or phenotype. Any genetic trait may be analyzed by the association method proposed here by carefully selecting the individuals to be included in the trait positive and trait negative phenotypic groups. Four criteria are often useful; clinical phenotype, age at onset, family history and severity. The selection procedure for continuous or quantitative traits (such as blood pressure for example) involves selecting individuals at opposite ends of the phenotype distribution of the trait under study, so as to include in these trait positive and trait negative populations individuals with overlapping phenotypes. Preferably, case-control populations consist of phenotypically homogeneous populations. Trait positive and trait negative populations consist of phenotypically uniform populations of individuals representing each between 1 and 98%, preferably between 1 and 80%, more preferably between 1 and 50%, and more preferably between 1 and 30%, most preferably between 1 and 20% of the total population under study, and preferably selected among individuals exhibiting non-overlapping phenotypes. The clearer the difference between the two trait phenotypes, the greater the probability of detecting an association with biallelic markers. The selection of those drastically different but relatively uniform phenotypes enables efficient comparisons in association studies and the possible detection of marked differences at the genetic level, provided that the sample sizes of the populations under study are significant enough.

In preferred embodiments, a first group of between 50 and 300 trait positive individuals, preferably about 100 individuals, are recruited according to their phenotypes. A similar number of control individuals are included in such studies.

In the present invention, typical examples of inclusion criteria include, but are not restricted to, prostate cancer or aggressiveness of prostate cancer tumors. In one preferred embodiment of the present invention, association studies are carried out on the basis of a presence (trait positive) or absence (trait negative) of prostate cancer.

Associations studies can be carried out by the skilled technician using the biallelic markers of the invention defined above, with different trait positive and trait negative populations. Suitable further examples of association studies using biallelic markers of the PCTA-1 gene, including the biallelic markers A1 to A125, involve studies on the following populations:

a trait positive population suffering from a cancer and a healthy unaffected population, or

a trait positive population suffering from prostate cancer treated with agents acting against prostate cancer and suffering from side-effects resulting from this treatment and an trait negative population suffering from prostate cancer treated with same agents without any substantial side-effects, or

a trait positive population suffering from prostate cancer treated with agents acting against prostate cancer showing a beneficial response and a trait negative population suffering from prostate cancer treated with same agents without any beneficial response, or

a trait positive population suffering from prostate cancer presenting highly aggressive prostate cancer tumors and a trait negative population suffering from prostate cancer with prostate cancer tumors devoid of aggressiveness.

Association Analysis

The general strategy to perform association studies using biallelic markers derived from a region carrying a candidate gene is to scan two groups of individuals (case-control populations) in order to measure and statistically compare the allele frequencies of the biallelic markers of the present invention in both groups.

If a statistically significant association with a trait is identified for at least one or more of the analyzed biallelic markers, one can assume that: either the associated allele is directly responsible for causing the trait (i.e. the associated allele is the trait causing allele), or more likely the associated allele is in linkage disequilibrium with the trait causing allele. The specific characteristics of the associated allele with respect to the candidate gene function usually give further insight into the relationship between the associated allele and the trait (causal or in linkage disequilibrium). If the evidence indicates that the associated allele within the candidate gene is most probably not the trait causing allele but is in linkage disequilibrium with the real trait causing allele, then the trait causing allele can be found by sequencing the vicinity of the associated marker, and performing further association studies with the polymorphisms that are revealed in an iterative manner.

Association studies are usually run in two successive steps. In a first phase, the frequencies of a reduced number of biallelic markers from the candidate gene are determined in the trait positive and control populations. In a second phase of the analysis, the position of the genetic loci responsible for the given trait is further refined using a higher density of markers from the relevant region. However, if the candidate gene under study is relatively small in length, as is the case for PCTA-1, a single phase may be sufficient to establish significant associations.

It is another object of the present invention to provide a method for the identification and characterization of an association between an allele of one or more biallelic markers of a PCTA-1 gene and a trait. The method comprises the steps of:

genotyping a marker or a group of biallelic markers according to the invention in trait positive and control individuals; and

establishing a statistically significant association between one allele of at least one marker and the trait.

The control individuals can be random or trait negative populations. Preferably, the trait positive and trait negative individuals are selected from non-overlapping phenotypes relating trait under study. In some embodiments, the biallelic marker is comprised in one or more of the sequences of P1 to P125, and the complementary sequences thereof.

The invention also comprises methods of detecting an association between a genotype and a phenotype, comprising the steps of a) determining the frequency of at least one PCTA-1-related biallelic marker in a trait positive population according to a genotyping method of the invention; b) determining the frequency of said PCTA-1-related biallelic marker in a control population according to a genotyping method of the invention; and c) determining whether a statistically significant association exists between said genotype and said phenotype. In addition, the methods of detecting an association between a genotype and a phenotype of the invention encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: Optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A44, A46 to A53, A57, A58, A62 to A76, A81, A82, A86 to A91, A107, A118, and A123 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A45, A54, A60, A61, A77 to A80, A83 to A85, A93, A102 to A106, A109, A110, A114, and A122, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A55, A56, A59, A92, A94 to A101, A108, A111 to A113, A115 to A117, and A119 to A121, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; Optionally, said control population may be a trait negative population, or a random population; Optionally, each of said genotyping steps a) and b) may be performed on a pooled biological sample derived from each of said populations; Optionally, each of said genotyping of steps a) and b) is performed separately on biological samples derived from each individual in said population or a subsample thereof; Optionally, the identity of the nucleotides at the biallelic markers of the PCTA-1 gene is determined in steps a) and b). Optionally, said phenotype is symptoms of, or susceptibility to cancer, preferably prostate cancer, the level of aggressiveness of prostate cancer tumors, an early onset of prostate cancer, a beneficial response to or side effects related to treatment against prostate cancer.

If the trait is a beneficial response or inversely a side effect to a treatment of prostate cancer, the method of the invention referred to above further comprises some or all of the following steps:

selecting a population or cohort of subjects diagnosed as suffering from prostate cancer;

administering a specified treatment of prostate cancer to said cohort of subjects;

monitoring the outcome of drug administration and identifying those individuals that are trait positive or trait negative relative to the treatment;

taking from said cohort biological samples containing DNA and testing this DNA for the presence of a specific allele or of a set of alleles of biallelic markers of the PCTA-1 gene;

analyzing the distribution of alleles of biallelic markers between trait positive and trait negative individuals; and

performing a statistical analysis to determine a statistically significant association between the presence or absence of alleles of biallelic markers of the PCTA-1 gene and the treatment related trait.

Haplotype Analysis

As described above, when a chromosome carrying a disease allele first appears in a population as a result of either mutation or migration, the mutant allele necessarily resides on a chromosome having a set of linked markers: the ancestral haplotype. This haplotype can be tracked through populations and its statistical association with a given trait can be analyzed. Complementing single point (allelic) association studies with multi-point association studies also called haplotype studies increases the statistical power of association studies. Thus, a haplotype association study allows one to define the frequency and the type of the ancestral carrier haplotype. A haplotype analysis is important in that it increases the statistical power of an analysis involving individual markers.

In a first stage of a haplotype frequency analysis, the frequency of the possible haplotypes based on various combinations of the identified biallelic markers of the invention is determined. The haplotype frequency is then compared for distinct populations of trait positive and control individuals. The number of trait positive individuals, which should be, subjected to this analysis to obtain statistically significant results usually ranges between 30 and 300, with a preferred number of individuals ranging between 50 and 150. The same considerations apply to the number of unaffected individuals (or random control) used in the study. The results of this first analysis provide haplotype frequencies in case-control populations, for each evaluated haplotype frequency a p-value and an odd ratio are calculated. If a statistically significant association is found the relative risk for an individual carrying the given haplotype of being affected with the trait under study can be approximated.

The present invention also provides a method for the identification and characterization of an association between a haplotype comprising alleles of several biallelic markers of the genomic sequence of the PCTA-1 gene and a trait. The method comprises the steps of:

genotyping a group of biallelic markers according to the invention in trait positive and control individuals; and

establishing a statistically significant association between a haplotype and the trait.

Preferably, the control individuals can be random or trait negative populations. In some embodiments, the haplotype comprises two or more biallelic markers comprised in the sequences of P1 to P125, and the complementary sequences thereof.

An additional embodiment of the present invention encompasses methods of detecting an association between a haplotype and a phenotype, comprising the steps of: a) estimating the frequency of at least one haplotype in a trait positive population, according to a method of the invention for estimating the frequency of a haplotype; b) estimating the frequency of said haplotype in a control population, according to a method of the invention for estimating the frequency of a haplotype; and c) determining whether a statistically significant association exists between said haplotype and said phenotype. In addition, the methods of detecting an association between a haplotype and a phenotype of the invention encompass methods with any further limitation described in this disclosure, or those following: Optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A44, A46 to A53, A57, A58, A62 to A76, A81, A82, A86 to A91, A107, A118, and A123 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A45, A54, A60, A61, A77 to A80, A83 to A85, A93, A102 to A106, A109, A110, A114, and A122, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A55, A56, A59, A92, A94 to A101, A108, A111 to A113, A115 to A117, and A119 to A121, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; Optionally, said control population is a trait negative population, or a random population. Optionally, said phenotype is symptoms of, or susceptibility to cancer, preferably prostate cancer, the level of aggressiveness of prostate cancer tumors, an early onset of prostate cancer, a beneficial response to or side effects related to treatment against prostate cancer; Optionally, said method comprises the additional steps of determining the phenotype in said trait positive and said control populations prior to step c).

Interaction Analysis

The biallelic markers of the present invention may also be used to identify patterns of biallelic markers associated with detectable traits resulting from polygenic interactions. The analysis of genetic interaction between alleles at unlinked loci requires individual genotyping using the techniques described herein. The analysis of allelic interaction among a selected set of biallelic markers with appropriate level of statistical significance can be considered as a haplotype analysis. Interaction analysis consists of stratifying the case-control populations with respect to a given haplotype for the first loci and performing a haplotype analysis with the second loci with each subpopulation.

Statistical methods used in association studies are further described below.

Testing for Linkage in the Presence of Association

The biallelic markers of the present invention may further be used in TDT (transmission/disequilibrium test). TDT tests for both linkage and association and is not affected by population stratification. TDT requires data for affected individuals and their parents or data from unaffected sibs instead of from parents (see Spielmann S. et al., 1993; Schaid D. J. et al., 1996, Spielmann S. and Ewens W. J., 1998). Such combined tests generally reduce the false—positive errors produced by separate analyses.

Association of Biallelic Markers of the Invention with Prostate Cancer

Trait Positive and Control Populations

Two groups of independent individuals were used: the overall trait positive and the trait negative populations included 491 individuals suffering from prostate cancer and 313 individuals without any sign of prostate cancer. A specific protocol for the collection of DNA samples from trait positive and trait negative individuals is described in Example 5. The 491 individuals suffering from prostate cancer can be subdivided into a population of individuals who developed prostate cancer under 65 years-old and a population of individuals who developed prostate cancer after the age of 65. The population of individuals who are less than 65 years-old was used to determine an association with an early onset of prostate cancer. The affected individuals can also be subdivided in familial cases and sporadic cases.

In order to have as much certainty as possible on the absence of prostate cancer in trait negative individuals, it is preferred to conduct a PSA dosage analysis on this population. Several commercial assays can be used (WO 96/21042, the disclosure of which is incorporated herein by reference in its entirety). In one preferred embodiment, a Hybritech assay is used and trait negative individuals must have a level of PSA less than 2.8 ng/ml of serum in order to be selected as such. In a preferred embodiment, the Yang assay is used and trait negative individuals must have a level of PSA of less than 4 ng/ml of serum in order to be included in the population under study.

Association Analysis

In one preferred embodiment of the invention in which a correlation was found between biallelic markers of the PCTA-1 gene and prostate cancer, results of the association study, further details of which are provided in example 5, seem to indicate that prostate cancer, preferably familial prostate cancer, more preferably early onset familial prostate cancer, is associated most strongly with the biallelic markers A30 (99-1572/440) and A41 (5-171/204) which present a particular interest. These association results constitute new elements for studying the genetic susceptibility of individuals to prostate cancer, preferably to familial prostate cancer, more preferably familial early onset prostate cancer. Further details concerning this association study are provided below.

The biallelic markers most strongly associated with prostate cancer, namely A30 and A41, are located in the regulatory region of the PCTA-1 gene, more particularly in the promoter region. The consequences of the presence of these markers in these regions are discussed below.

Furthermore, the biallelic marker A2 (99-1601/402) was found to be also associated with prostate cancer, more particularly with sporadic prostate cancer. This biallelic marker is localized in the 5′ regulatory region of the PCTA-1 gene.

Similar association studies can also be carried out with other biallelic markers within the scope of the invention, preferably with biallelic markers in linkage disequilibrium with the markers associated with prostate cancer as described above, including the biallelic markers A1 to A125.

Analysis of Biallelic Marker Associations

Even though polymorphisms associated with prostate cancer have been identified in the coding region of the PCTA-1 gene, these polymorphisms do not appear to be as significant as those found in the upstream regulatory region of the PCTA-1 gene. The results further suggest that a trait-causing mutation is likely to be located within the 5′ regulatory region of the PCTA-1 gene. The extent to which the markers found within the coding region of PCTA-1 are significant in relation to cancer can be determined using haplotype analyses involving at least two of the biallelic markers of the present invention.

Six of the biallelic markers of the present invention result in a change in the amino acid sequence of a PCTA-1 protein. These are biallelic markers A54, A56, A60, A75, A76 and A85. These mutations may change the function and/or the stability of the PCTA-1 protein. An amino acid change in a PCTA-1 protein can lead to alterations in PCTA-1 biological activity. Either a modified function or an increased stability can be involved in prostate cancer appearance.

Furthermore, as the expression of the PCTA-1 gene has mainly been reported in prostate cancer cells, one can assume that its expression is closely linked to the development of cancer, particularly prostate cancer. Generally, a major control of gene expression proceeds at the level of the initiation of the transcription. This initiation involves the promoter which can be considered as a concentration of transcription factor binding sites. The initiation of the transcription also involves enhancers which modulate the efficiency of the initiation and consist of DNA binding sites which are located in regulatory regions of the considered gene which may be at a certain distance in 3′ or 5′ of the gene.

Most of the biallelic polymorphisms of the PCTA-1 gene associated with prostate cancer according to the present invention are located in the regulatory region upstream of the transcription start site of the PCTA-1 gene and particularly in the promoter. Biallelic marker A41, which is located about 120 bp upstream of the beginning of the first exon (exon 0), may be comprised in the proximal promoter of the PCTA-1 gene. This biallelic marker could be a trait causing mutation of prostate cancer. Biallelic marker A30, which is located about 1.5 kb upstream the beginning of the first exon (exon 0), may be comprised in the distal promoter of the PCTA-1 gene. Biallelic marker A2 is located in the 5′ regulatory region of the PCTA-1 gene.

As the expression of the PCTA-1 gene has mainly been reported in prostate cancer cells, the expression of PCTA-1 gene is modified during the carcinogenesis. The exact mechanism through which PCTA expression is modified is not understood. However, it is possible that the polymorphisms A41, A30, and A2 modulate PCTA-1 expression by modulating PCTA-1 transcription through DNA binding proteins, which will be explained in further detail below.

The regulation of PCTA-1 expression is a key factor in the onset and for development of cancer and particularly prostate cancer. In this regard, the polymorphisms located in the 5′ regulatory region of the PCTA-1 gene appear to play the most significant role in the association of PCTA-1 with cancer. It appears clear that the polymorphisms found in the promoter region adjacent to the transcription initiation site, and particularly those located in the proximal PCTA-1 promoter, are more strongly associated with prostate cancer than polymorphisms of the other promoter elements located further upstream of this site. Furthermore, some polymorphisms, such as the biallelic marker A41, are clearly associated with early onset prostate cancer. The polymorphisms found in the proximal 2000 to 3000 bp of the 5′ regulatory region are associated with early onset prostate cancer. The inventors have also shown an association between some of the biallelic markers of the present invention located at the 3′ end of the PCTA-1 genomic DNA and prostate cancer.

The involvement of the associated polymorphisms in the modification of the PCTA-1 expression in prostate cancer cells can be confirmed through the assays described below.

The expression levels of a PCTA-1 gene, preferably a gene comprising at least one biallelic marker according to the invention, in different tissues, can be determined by analyses of tissue samples from individuals typed for the presence or absence of a specific polymorphism. Any convenient method can be used such as Northern, or Dot blot or other hybridization analyses, and quantitative RT-PCR for mRNA quantitation, Western blot ELISA, R1A for protein quantitation. The tissue specific expression can then be correlated with the genotype. More details on some of these methods are provided below under the heading “Method For Screening”.

The effects of modifications in the regulatory regions of the PCTA-1 gene, and particularly in the sequence of its promoter, can be studied through the determination of expression levels by expression assays for the particular promoter sequence. The assays are performed with the PCTA-1 coding sequence or with a detectable marker sequence using a reporter gene. To determine tissue specificity, the assay is performed in cells from different sources. Preferably the assay is performed on normal tissue cells and cancerous cells of the same tissue type (e.g. prostate cells and on prostate cancer cells). More preferably, the assay is performed on a large range of cell lines with an increasing level of malignancy. Some methods are discussed in more detail below under the heading “Method For Screening”.

An assay to determine the effect of a sequence polymorphism on PCTA-1 expression may be performed in cell-free extracts, or in cellculture assays, such as transient or stable transfection assays. This assay is also within the scope of the present invention. Alterations in expression may be correlated to decreases or increases in the basic amounts of PCTA-1 mRNA and/or protein that are expressed in one or more cell types. Expression levels of different alleles are compared using various methods known in the art. Methods for determining whether the level of expression triggered by promoter or enhancer sequences is increased or decreased depending on the studied allele of said sequence include the insertion into a vector of said sequence upstream a reporter gene such as β-galactosidase, luciferase, green fluorescent protein or chloramphenicol acetyltransferase. Expression levels are assessed by quantitation of expressed reporter proteins that provides for convenient quantitation.

The changes in PCTA-1 expression can be the result of modifications in the modulation of PCTA-1 transcription by DNA binding proteins, which are able to activate or inhibit the initiation of the transcription of the PCTA-1 gene. The term “DNA binding protein” is intended to encompass more particularly transcriptional factors. The binding of these proteins on the sites located in the promoter is critical for a correct binding of polymerases and consequently for the initiation of transcription. The binding of these proteins on the sites located in the 5′ upstream regulatory regions modulates transcription.

The binding sites of DNA binding proteins, preferably transcription factors, are generally 6-20 nucleotides in length. A polymorphic site located in a transcription factor binding site may result in a difference of binding affinity of the said transcription factor between the two allele of the polymorphism. This difference of affinity could explain the changes of expression of the PCTA-1 gene.

When one or more alleles of the biallelic markers of the PCTA-1 gene associated with cancer are present in the genome of an individual since conception, there would be an event which provokes a drastic increase in the expression of PCTA-1. There are at least two possible hypotheses that can be formulated to explain this event. Firstly, as cancer is the result of a succession of mutations, one mutation could lead to either the expression of a new DNA binding activity, or the overexpression of a DNA binding factor which binds to the site containing the polymorphism and which is involved in the transcription of the PCTA-1 gene. Secondly the DNA binding factor readily binds to the site containing the polymorphism in normal cells where it is either unable to activate the transcription of PCTA-1 or repressor of the PCTA-1 transcription initiation. A mutation in the transcription factor can make the transcription factor either functional in the case of an activator or unfunctional in the case of a repressor. Likewise, a mutation in an additional protein can induce the binding of this protein which is needed by the DNA binding factor for activating the transcription of the PCTA-1 gene.

In order to confirm the capacity of transcription factors to bind sites containing the biallelic markers of the present invention, so as to assess the difference in affinity between the two alleles of the considered biallelic marker and to discriminate between these hypotheses, a gel retardation assay or DNA mobility shift assay can be carried out. This type of assay is well-known to those skilled in the art and is described in U.S. Pat. No. 5,698,389, U.S. Pat. No. 5,502, 176, Fried and Crothers (1981), Garner and Revzin (1981) and Dent and Latchman (1993).

This type of method relies on the principle that a fragment of DNA to which a protein has bound will move more slowly in gel electrophoresis than the same DNA fragment without the bound protein. The DNA mobility shift assay is carried out, therefore, by first labeling the specific DNA segment whose protein-binding properties are being investigated. The labeled DNA is then incubated with a nuclear (Dignam et al., 1983; Schreiber et al., 1989; Mulleret al., 1989; Mizokami et al., 1994) or whole cell (Manley et al., 1980) extract of cells prepared in such a way as to contain DNA-binding proteins. DNA-protein complexes are then allowed to form. The complexes are then electrophoresed on a non-denaturing polyacrylamide gel and the position of the labeled DNA is visualized by suitable techniques. Various types of suitable labels can be selected by the person skilled in the art. Notably, the radioactive labeling is appropriate. If no protein has bound to the DNA, all the label is free to migrate quickly, whereas labeled protein-DNA complexes migrate more slowly and hence give a different signal from that of the unbound DNA near the top of the gel. The interaction specificity can be estimated by carrying out a gel retardation assay with increasing amount of unlabeled DNA segment which can compete with the labeled one. A positive control can be realized with an oligonucleotide containing the androgene responsive element.

The investigated DNA segment preferably comprises the sequence of a potential binding site containing an allele of a polymorphism of the present invention, more preferably a sequence comprising a sequence selected from P1 to P125 and the complementary sequences thereto, still more preferably a sequence comprising a sequence selected from P1 to P43 and the complementary sequences thereto. In an embodiment, the polymorphism site is located in the middle of the DNA fragment. In an other embodiment, the polymorphism site can be located close to an end of the DNA fragment, for example at 6 nucleotides away from the end. The DNA fragment has a sufficient length to hybridize with the complementary strand and to form a stable double strand. For example, the DNA fragment comprises at least 8 nucleotides, preferably at least 20 nucleotides, more preferable 30 nucleotides. In a specific embodiment, the DNA fragment comprises the sequence of interest at the middle of the fragment and some poly G, poly C, or poly GC at its 5′ and/or 3′ ends.

In a preferred embodiment, the DNA segment consists of an oligonucleotide selected from the group consisting of Oligo1 to Oligo60 which are described in Table C and detailed as feature in SEQ ID No 1. For each polymorphic site, 4 oligonucleotides are generated and correspond to the two complementary strands of the DNA for each of the two alleles of the considered polymorphism. The DNA segments are designed such as the polymorphic base is surrounded with 14 nucleotides on each side.

TABLE C Complementary Position range of the position range of the Oligo- oligonucleotide Oligo- oligonucleotide in SEQ Biallelic nucleotide in SEQ ID No 1 nucleotide ID No 1 marker All name Beginning End name Beginning End 5-169-208 A Oligo1  67820 67848 Oligo31 67820 67850 5-169-208 G Oligo2  67820 67848 Oligo32 67820 67850 5-169-331 C Oligo3  67940 67969 Oligo33 67941 67969 5-169-331 T Oligo4  67940 67969 Oligo34 67941 67969 5-169-97 C Oligo5  67707 67737 Oligo35 67709 67738 5-169-97 G Oligo6  67707 67737 Oligo36 67709 67738 5-170-238 A Oligo7  68198 68227 Oligo37 68199 68228 5-170-238 G Oligo8  68198 68227 Oligo38 68199 68228 5-170-288 A Oligo9  68247 68277 Oligo39 68249 68277 5-170-288 C Oligo10 68247 68277 Oligo40 68249 68277 5-171-156 G Oligo11 68463 68491 Oligo41 68463 68492 5-171-156 T Oligo12 68463 68491 Oligo42 68463 68492 5-171-204 C Oligo13 68511 68539 Oligo43 68511 68539 5-171-204 T Oligo14 68511 68539 Oligo44 68511 68539 5-171-273 A Oligo15 68580 68608 Oligo45 68580 68608 5-171-273 G Oligo16 68580 68608 Oligo46 68580 68608 5-171-289 C Oligo17 68596 68624 Oligo47 68596 68626 5-171-289 T Oligo18 68596 68624 Oligo48 68596 68626 5-171-54 C Oligo19 68360 68389 Oligo49 68361 68389 5-171-54 G Oligo20 68360 68389 Oligo50 68361 68389 99-1572-315 C Oligo21 66951 66981 Oligo51 66953 66983 99-1572-315 T Oligo22 66951 66981 Oligo52 66953 66983 99-1572-335 A Oligo23 66973 67001 Oligo53 66973 67002 99-1572-335 G Oligo24 66973 67001 Oligo54 66973 67002 99-1572-440 C Oligo25 67078 67106 Oligo55 67078 67106 99-1572-440 T Oligo26 67078 67106 Oligo56 67078 67106 99-1572-477 A Oligo27 67113 67143 Oligo57 67115 67144 99-1572-477 T Oligo28 67113 67143 Oligo58 67115 67144 99-1572-578 C Oligo29 67212 67243 Oligo59 67215 67247 99-1572-578 T Oligo30 67212 67243 Oligo60 67215 67247

Each oligonucleotide selected from Oligo1 to Oligo60 comprises 4 additional bases, namely GATC, at its 5′ end.

In a preferred embodiment, either the nuclear or whole cell extracts are provided from normal and cancer cells, particularly from normal prostate cells and prostate cancer cells. For example, suitable cell extracts can be provided from PZ-HPV-7 (ATCC: CRL-222 1), CA-HPV-10 (ATCC CRL-2220), PC-3 (ATCC: CRL-1435), DU 145 (ATCC: HTB-81), LNCaP-FGC (ATCC: CRL-10995 and CRL-1740), or NCI-H660 (ATCC: CRL-5813) cells. In a more preferred embodiment, the cell extracts are provided form PNT1A, PNT2, LNCaP-JMV, DU145 (ATCC Nr: HTB-81) or PC3 (ATCC Nr: CRL-1435) cells.

In case a new transcription factor is specifically expressed in cancer cells, a gel retardation assay will show a retarded or shifted band only when the DNA was incubated with cell extracts from prostate cancer cells. If the DNA binding activity already exists in normal cells, the gel retardation assay will show a shifted band with cell extracts from normal prostate cells and prostate cancer cells. Gel retardation assays will also allow to show a significant difference in affinity between a DNA binding factor and binding sites containing the two alleles of the considered polymorphism.

The interaction of the DNA segment described above with transcription factors can also be studied with an optical biosensor such as BIACORE. This technology is well-known to those skilled in the art and is described in Szabo et al. (1995) and Edwards et al. (1997). The main advantage of this method is that it allows the determination of the association rate between the DNA fragment which is investigated and the DNA binding protein. Typically, a DNA segment such as those defined above is biotinylated at its 5′ or 3′ ends and is immobilized on a streptavidin-coated sensor chip. Then, a whole or a nuclear extract of cells is placed in contact with the DNA segment. The binding of DNA binding proteins to the DNA fragment causes a change in the refractive index and/or thickness. This change is detected by the Biosensor provided it occurs in the evanescent field. The affinity of the DNA binding protein to the DNA fragment can then be measured.

In order to precisely localize the binding site of the transcription factors, DNAse I footprinting or DMS protection footprinting assays can also be carried out with DNA fragments which contain the sequence of a potential binding site containing an allele of a polymorphism of the present invention, preferably a sequence comprising a sequence selected from P1 to P125 and the complementary sequences thereto, more preferably a sequence comprising a sequence selected from P1 to P43 and the complementary sequences thereto. This type of assay is well-known to those skilled in the art and is described in Galas and Schmitz (1978), and Dynan and Tjian (1 983). Briefly, in the DNAse I footprinting assay, end-labeled DNA is incubated with protein extract and then partially digested with DNAse I. Specific binding of proteins to DNA will modify nuclease digestion at the site of interaction relative to free DNA, leaving an “imprint” which can be visualized after extraction of the labeled DNA and electrophoresis in a sequence gel.

The interaction with transcription factors can also be studied with the methylation interference assay which is well-known to those skilled in the art and is described in Siebenlist and Gilbert (1980) and Maxam and Gilbert (1980). Briefly, this method relies on the ability of DMS to methylate G residues, which can be cleaved with piperidine. The target DNA is partially methylated so that, on average, only one G residue per DNA molecule is methylated. These partially methylated molecules is used in a DNA mobility shift experiment with an appropriate cell extract containing transcription factors. After electrophoresis, the band produced by the DNA which has bound protein and that produced by the unbound DNA are excised from the gel and treated with piperidine to cleave the DNA at the methylated G residues and not at unmethylated G residues. If methylation of a particular G residue prevents transcription factors binding, then cleavage at this methylated G residue will be observed only in the DNA that failed to bind the protein.

In order to confirm the implication of a particular PCTA-1 derived sequence containing the biallelic marker as a binding site for a transcription regulator of PCTA-1 in cancer cells, a transient expression assay can be carried out in which a vector comprising the considered binding site upstream of the HSV1 thymidine kinase promoter operably linked to a reporter gene such as chloramphenicol acetyltransferase is transfected in appropriate cell lines. This assay is well-known to those skilled in the art and is described in Doucas et al. (1991). This assay can also be realized by cloning the considered binding site upstream the SV40 promoter into the pGL3-promoter luciferase vector (Promega) as described in Coles et al.(1998). Both normal and cancer cells, more particularly normal and cancer cells from prostate, are transfected with said vector. The effect of the binding site and more particularly of the alleles comprised in the binding site can be assessed through the expression level of the reporter gene.

The inventors believe that these polymorphisms, particularly the polymorphisms located on or close to polyadenylation sites have a direct although somewhat milder effect on prostate cancer development.

Haplotype Analysis

In the context of the present invention, a haplotype can be defined as a combination of biallelic markers found in a given individual and which may be associated more or less significantly, as a result of appropriate statistical analyses, with the expression of a given trait.

A two-marker haplotype including markers A30 and A41 (TT alleles respectively) was shown to be significantly associated with prostate cancer, preferably with a familial prostate cancer, more preferably with a familial early onset prostate cancer. As shown in Table 8, the “TT” haplotype present a p-value of 2.5×10⁻⁶ for the familial early onset prostate cancer (see Example 5).

A three-marker haplotype including markers A2, A30, and A41 (ATT alleles respectively) was shown to be significantly associated with prostate cancer, preferably with a familial prostate cancer, more preferably with a familial early onset prostate cancer. As shown in table 8, the “ATT” haplotype present a p-value of 2.5×10⁻⁷ for the familial early onset prostate cancer (see Example 5).

A first two-marker haplotype including markers A2 and A57 (99-1605/112) (TA alleles, respectively) was shown to be significantly associated with prostate cancer, preferably with a sporadic prostate cancer. As shown in table 8, the “TA” haplotype present a p-value of 3.4×10⁻⁵ for the sporadic informative prostate cancer (see Example 5). A second two-marker haplotype including markers A2 and A55 (5-2/178) (TT alleles, respectively) was shown to be significantly associated with prostate cancer, preferably with a sporadic prostate cancer. As shown in table 8, the “TT” haplotype present a p-value of 1×10⁻⁵ for the sporadic informative prostate cancer (see Example 5).

Therefore, one preferred haplotype of the present invention associated with a familial prostate cancer comprises a biallelic marker selected from the group consisting of A30 (allele T), A41 (allele T), A2 (allele A), A55 (allele C) and A57 (allele G). One more preferred haplotype of the present invention associated with a familial prostate cancer comprises a biallelic marker selected from the group consisting of A30 (allele T), A41 (allele T), and A2 (allele A). One still more haplotype of the present invention associated with a familial prostate cancer comprises a biallelic marker selected from the group consisting of A30 (allele T), and A41 (allele T).

Furthermore, one preferred haplotype of the present invention associated with a sporadic prostate cancer comprises a biallelic marker selected from the group consisting of A2 (allele T), A55 (allele T), A57 (allele A), A30 (allele T) and A41 (allele T). One more preferred haplotype of the present invention associated with a sporadic prostate cancer comprises a biallelic marker selected from the group consisting of A2 (allele T), A41 (allele T), A55 (allele T), A57 (allele A).

The permutation tests clearly validated the statistical significance of the association between these haplotypes and the prostate cancer (see Example 5). All these haplotypes can be used in diagnostic of prostate cancer, more particularly either familial prostate cancer or sporadic prostate cancer.

One can observe that the haplotypes associated to familial cases of prostate cancer are not associated with the sporadic cases of prostate cancer and that the haplotypes associated to the sporadic cases are not associated with the familial cases (see Table 7 of Example 5). Moreover, except the biallelic markers A2, the familial and sporadic cases haplotypes do not present any common biallelic marker. Therefore, the ancestral haplotypes would be different and the causing trait allele would not be the same.

This information is extremely valuable. The knowledge of a potential genetic predisposition to prostate cancer, even if this predisposition is not absolute, might contribute in a very significant manner to treatment efficacy of prostate cancer and to the development of new therapeutic and diagnostic tools.

Statistical Methods

In general, any method known in the art to test whether a trait and a genotype show a statistically significant correlation may be used.

1) Methods in Linkage Analysis

Statistical methods and computer programs useful for linkage analysis are well-known to those skilled in the art (see Terwilliger J. D. and Ott J., 1994; Ott J., 1991).

2) Methods to Estimate Haplotype Frequencies in a Population

As described above, when genotypes are scored, it is often not possible to distinguish heterozygotes so that haplotype frequencies cannot be easily inferred. When the gametic phase is not known, haplotype frequencies can be estimated from the multilocus genotypic data. Any method known to person skilled in the art can be used to estimate haplotype frequencies (see Lange K., 1997; Weir, B. S., 1996) Preferably, maximum-likelihood haplotype frequencies are computed using an Expectation- Maximization (EM) algorithm (see Dempster et al., 1977; Excoffier L. and Slatkin M., 1995). This procedure is an iterative process aiming at obtaining maximum-likelihood estimates of haplotype frequencies from multi-locus genotype data when the gametic phase is unknown. Haplotype estimations are usually performed by applying the EM algorithm using for example the EM-HAPLO program (Hawley M. E. et al., 1994) or the Arlequin program (Schneider et al., 1997). The EM algorithm is a generalized iterative maximum likelihood approach to estimation and is briefly described below.

Please note that in the present section, “Methods To Estimate Haplotype Frequencies In A Population,” of this text, phenotypes will refer to multi-locus genotypes with unknown phase. Genotypes will refer to known-phase multi-locus genotypes.

A sample of N unrelated individuals is typed for K markers. The data observed are the unknown-phase K-locus phenotypes that can categorized in F different phenotypes. Suppose that we have H underlying possible haplotypes (in case of K biallelic markers, H=2^(K)).

For phenotype j, suppose that c_(j) genotypes are possible. We thus have the following equation $\begin{matrix} {P_{j} = {{\sum\limits_{i = 1}^{c_{j}}{{pr}\left( {genotype}_{i} \right)}} = {\sum\limits_{i = 1}^{c_{j}}{{pr}\left( {h_{k},h_{l}} \right)}}}} & {{Equation}\quad 1} \end{matrix}$

where P_(j) is the probability of the phenotype j, h_(k) and h_(l) are the two haplotypes constituent the genotype i. Under the Hardy-Weinberg equilibrium, pr(h_(k), h_(l)) becomes:

pr(h _(k), h_(l))=pr(h _(k))²if h _(k) =h _(l) , pr(h _(k) , h _(l))=2pr(h _(k)).pr(h _(l)) if h _(k) ≠h _(l).  Equation 2

The successive steps of the E-M algorithm can be described as follows:

Starting with initial values of the of haplotypes frequencies, noted p₁ ⁽⁰⁾, p₂ ⁽⁰⁾, . . . p_(H) ⁽⁰⁾, these initial values serve to estimate the genotype frequencies (Expectation step) and then estimate another set of haplotype frequencies (Maximization step), noted p₁ ⁽¹⁾, p₂ ⁽¹⁾, . . . p_(H) ⁽¹⁾, these two steps are iterated until changes in the sets of haplotypes frequency are very small.

A stop criterion can be that the maximum difference between haplotype frequencies between two iterations is less than 10⁻⁷. These values can be adjusted according to the desired precision of estimations.

At a given iteration s, the Expectation step consists of calculating the genotypes frequencies by the following equation: $\begin{matrix} \begin{matrix} {{{pr}\left( {genotype}_{i} \right)}^{(s)} = \quad {{{pr}\left( {phenotype}_{j} \right)} \cdot}} \\ \left. {{\quad {{pr}\left( {genotype}_{i} \right.}}{phenotype}_{j}} \right)^{(s)} \\ {= \quad {\frac{n_{j}}{N} \cdot \frac{{{pr}\left( {h_{k},h_{l}} \right)}^{(s)}}{P_{j}^{(s)}}}} \end{matrix} & {{Equation}\quad 3} \end{matrix}$

where genotype i occurs in phenotype j, and where h_(k) and h_(l) constitute genotype i. Each probability is derived according to eq. 1, and eq. 2 described above.

Then the Maximization step simply estimates another set of haplotype frequencies given the genotypes frequencies. This approach is also known as the gene-counting method (Smith, 1957). $\begin{matrix} {p_{t}^{({s + 1})} = {\frac{1}{2}{\sum\limits_{j = 1}^{F}{\sum\limits_{i = 1}^{c_{j}}{\delta_{it} \cdot {{pr}\left( {genotype}_{i} \right)}^{(s)}}}}}} & {{Equation}\quad 4} \end{matrix}$

Where δ_(it) is an indicator variable which count the number of time haplotype t in genotype i. It takes the values of 0, 1 or 2.

To ensure that the estimation finally obtained is the maximum-likelihood estimation several values of departures are required. The estimations obtained are compared and if they are different the estimations leading to the best likelihood are kept.

3) Methods to Calculate Linkage Disequilibrium Between Markers

A number of methods can be used to calculate linkage disequilibrium between any two genetic positions, in practice linkage disequilibrium is measured by applying a statistical association test to haplotype data taken from a population.

Linkage disequilibrium between any pair of biallelic markers comprising at least one of the biallelic markers of the present invention (M_(i), M_(j)) having alleles (a_(i)/b_(i)) at marker M_(i) and alleles (a_(j)/b_(j)) at marker M_(j) can be calculated for every allele combination (a_(i),a_(j), a_(i),b_(j): b_(i),a_(j) and b_(i),b_(j)), according to the Piazza formula:

Δ_(aiaj)=θ4−(θ4+θ3) (θ4+θ2), where:

θ4=−−=frequency of genotypes not having allele a_(i) at M_(i) and not having allele a_(j) at M_(j)

θ3=−+=frequency of genotypes not having allele a_(i) at M_(i) and having allele a_(j) at M_(j)

θ2=+−=frequency of genotypes having allele a_(i) at M_(i) and not having allele a_(j) at M_(j)

Linkage disequilibrium (LD) between pairs of biallelic markers (M_(i); M_(j)) can also be calculated for every allele combination (ai,aj; ai,bj; b_(i),a_(j) and b_(i),b_(j)), according to the maximum-likelihood estimate (MLE) for delta (the composite genotypic disequilibrium coefficient), as described by Weir (Weir B. S., 1996). The MLE for the composite linkage disequilibrium is:

D _(aiaj) (2n ₁ +n ₂ +n ₃ +n ₄/2)/N−2(pr(a _(i)). pr(a _(j)))

Where n₁=Σ phenotype (a_(i)/a_(i),a_(j)/a_(j)), n₂=Σ phenotype (a_(i)/a_(i), a_(j)/b_(j)), n₃=Σ phenotype (a_(i),/b_(i), a_(j)/a_(j)), n₄=Σ phenotype (a_(i)/b_(i), a_(j)/b_(j)) and N is the number of individuals in the sample.

This formula allows linkage disequilibrium between alleles to be estimated when only genotype, and not haplotype, data are available.

Another means of calculating the linkage disequilibrium between markers is as follows. For a couple of biallelic markers, M_(i) (a_(i)/b_(i)) and M_(j)(a_(j)/b_(j)), fitting the Hardy-Weinberg equilibrium, one can estimate the four possible haplotype frequencies in a given population according to the approach described above.

The estimation of gametic disequilibrium between ai and aj is simply:

D _(aiaj) =pr(haplotype(a _(i) ,a _(j)))−pr(a _(i)).pr(a _(j)).

Where pr(a_(i)) is the probability of allele a_(i) and pr(a_(i)) is the probability of allele a_(j) and where pr(haplotype (a_(i), a_(j))) is estimated as in Equation 3 above.

For a couple of biallelic marker only one measure of disequilibrium is necessary to describe the association between M_(i) and M_(j).

Then a normalized value of the above is calculated as follows:

D′_(aiaj) =D _(aiaj)/max (−pr(a _(i)). pr(a_(j)), −pr(b _(i)). pr(b _(j))) with D _(aiaj)<0

D′_(aiaj) =D _(aiaj)/max (pr(b _(i)). pr(a_(j)), pr(a _(i)). pr(b _(j))) with D _(aiaj)>0

The skilled person will readily appreciate that other linkage disequilibrium calculation methods can be used.

Linkage disequilibrium among a set of biallelic markers having an adequate heterozygosity rate can be determined by genotyping between 50 and 1000 unrelated individuals, preferably between 75 and 200, more preferably around 100.

4) Testing for Association

Methods for determining the statistical significance of a correlation between a phenotype and a genotype, in this case an allele at a biallelic marker or a haplotype made up of such alleles, may be determined by any statistical test known in the art and with any accepted threshold of statistical significance being required. The application of particular methods and thresholds of significance are well with in the skill of the ordinary practitioner of the art.

Testing for association is performed by determining the frequency of a biallelic marker allele in case and control populations and comparing these frequencies with a statistical test to determine if their is a statistically significant difference in frequency which would indicate a correlation between the trait and the biallelic marker allele under study. Similarly, a haplotype analysis is performed by estimating the frequencies of all possible haplotypes for a given set of biallelic markers in case and control populations, and comparing these frequencies with a statistical test to determine if their is a statistically significant correlation between the haplotype and the phenotype (trait) under study. Any statistical tool useful to test for a statistically significant association between a genotype and a phenotype may be used. Preferably the statistical test employed is a chi-square test with one degree of freedom. A P-value is calculated (the P-value is the probability that a statistic as large or larger than the observed one would occur by chance).

Statistical Significance

In preferred embodiments, significance for diagnosis purposes, either as a positive basis for further diagnostic tests or as a preliminary starting point for early preventive therapy, the p value related to a biallelic marker association is preferably about 1×10⁻² or less, more preferably about 1×10⁻⁴ or less, for a single biallelic marker analysis and about 1×10⁻³ or less, still more preferably 1×10⁻⁶ or less and most preferably of about 1×10⁻⁸ or less, for a haplotype analysis involving two or more markers. These values are believed to be applicable to any association studies involving single or multiple marker combinations.

The skilled person can use the range of values set forth above as a starting point in order to carry out association studies with biallelic markers of the present invention. In doing so, significant associations between the biallelic markers of the present invention and prostate cancer, the level of aggressiveness of prostate cancer tumors, an early onset of prostate cancer, or a beneficial response to or side effects related to treatment against prostate cancer can be revealed and used for diagnosis and drug screening purposes.

Phenotypic Permutation

In order to confirm the statistical significance of the first stage haplotype analysis described above, it might be suitable to perform further analyses in which genotyping data from case-control individuals are pooled and randomized with respect to the trait phenotype. Each individual genotyping data is randomly allocated to two groups, which contain the same number of individuals as the case-control populations used to compile the data obtained in the first stage. A second stage haplotype analysis is preferably run on these artificial groups, preferably for the markers included in the haplotype of the first stage analysis showing the highest relative risk coefficient. This experiment is reiterated preferably at least between 100 and 10000 times. The repeated iterations allow the determination of the probability to obtain by chance the tested haplotype.

Assessment of Statistical Association

To address the problem of false positives similar analysis may be performed with the same case-control populations in random genomic regions. Results in random regions and the candidate region are compared as described in a co-pending US Provisional Patent Application entitled “Methods, Software And Apparati For Identifying Genomic Regions Harboring A Gene Associated With A Detectable Trait,” U.S. Ser. No. 60/107,986, filed Nov. 10, 1998.

5) Evaluation of Risk Factors

The association between a risk factor (in genetic epidemiology the risk factor is the presence or the absence of a certain allele or haplotype at marker loci) and a disease is measured by the odds ratio (OR) and by the relative risk (RR). If P(R⁺) is the probability of developing the disease for individuals with R and P(R⁻) is the probability for individuals without the risk factor, then the relative risk is simply the ratio of the two probabilities, that is:

RR=P(R ⁺)/P(R ⁻)

In case-control studies, direct measures of the relative risk cannot be obtained because of the sampling design. However, the odds ratio allows a good approximation of the relative risk for low-incidence diseases and can be calculated:

OR=(F ⁺/(1−F ⁺))/(F ⁻/(1−F ⁻))

F⁺ is the frequency of the exposure to the risk factor in cases and F⁻ is the frequency of the exposure to the risk factor in controls. F⁺ and F⁻ are calculated using the allelic or haplotype frequencies of the study and further depend on the underlying genetic model (dominant, recessive, additive . . . ).

One can further estimate the attributable risk (AR) which describes the proportion of individuals in a population exhibiting a trait due to a given risk factor. This measure is important in quantifying the role of a specific factor in disease etiology and in terms of the public health impact of a risk factor. The public health relevance of this measure lies in estimating the proportion of cases of disease in the population that could be prevented if the exposure of interest were absent. AR is determined as follows:

AR=P _(E)(RR−1)/(P _(E)(RR−1 )+1)

AR is the risk attributable to a biallelic marker allele or a biallelic marker haplotype. P_(E) is the frequency of exposure to an allele or a haplotype within the population at large; and RR is the relative risk which, is approximated with the odds ratio when the trait under study has a relatively low incidence in the general population.

Identification of Biallelic Markers in Linkage Disequilibrium with the PCTA-1-Related Biallelic Markers

Once an association has been demonstrated between a given biallelic marker and a trait, the discovery of additional biallelic markers associated to trait and in linkage disequilibrium with one of the biallelic markers disclosed herein can easily be carried out by the skilled person.

The present invention then also concerns biallelic markers in linkage disequilibrium with the specific biallelic markers described above, more particularly with biallelic markers Al to A125, and which are expected to present similar characteristics in terms of their respective association with a given trait.

Hence, once linkage disequilibrium has been demonstrated between a trait and a given biallelic marker, all the biallelic markers shown to be in linkage disequilibrium with the given biallelic marker are expected to present similar characteristics in terms of their respective association with a given trait. The discovery of additional biallelic markers associated with this trait is of great interest in order to increase the density of biallelic markers in this particular region because the causal mutation will be found in the vicinity of the marker or set of markers showing the highest correlation with the trait. These additional markers which can be identified and sequenced by the skilled person using the teachings of the present application also fall within the scope of the present invention.

The invention also concerns a method for the identification and characterization of a biallelic marker in linkage disequilibrium with a biallelic marker of the PCTA-1 gene, preferably a biallelic marker of the PCTA-1 gene of which one allele is associated with a trait In one embodiment, the biallelic marker of the PCTA-1 gene is outside of the PCTA-1 gene itself. In another embodiment, the biallelic marker in linkage disequilibrium with a biallelic marker of the PCTA-1 gene is itself located within the PCTA-1 gene. The method comprises the following steps: (a) amplifying a genomic fragment, preferably comprising a first biallelic marker, from a plurality of individuals; (b) identifying second biallelic markers in said amplified portion; (c) conducting a linkage disequilibrium analysis between said first biallelic marker and second biallelic markers; and, (d) identifying second biallelic markers in linkage disequilibrium with said first marker. Subcombinations comprising steps (b) and (c) are also contemplated. Optionally, the first biallelic marker is selected from the group consisting of A1 to A125 and the complements thereof. Preferably, the first biallelic marker is selected from the group consisting of A2, A30, A41, A55, A57 and the complements thereof.

Methods to identify biallelic markers and to conduct linkage disequilibrium analysis are described herein and can be carried out by the skilled person without undue experimentation.

Once identified, the sequences in linkage disequilibrium with a biallelic marker of the PCTA-1 gene may be used in any of the methods described herein, including methods for determining an association between a biallelic marker and a trait, methods for identifying individuals having a predisposition for a trait, methods of administration of prophylactic or therapeutic agents disease treatment, methods of identifying individuals likely to respond positively or negatively to said agents, and methods of using drugs and vaccines.

An example of identification of additional biallelic markers associated to a trait based on the previous knowledge of the localization of a first marker associated to a given trait is given below.

Biallelic Markers in Linkage Disequilibrium with a Particular Marker:Apo E4

The following example relating to the identification of markers in linkage disequilibrium with the apoE4 allele is representative of the procedures of the present invention in which markers in LD with a target gene are identified. 3 major isoforms of human apolipoprotein E (apoE2, -E3, and -E4) have been identified by isoelectric focusing and are coded for by 3 alleles (ε2, 3, and 4) of the Apo E gene. As originally reported by Strittrnatter et al. and by Saunders et al. in 1993, the Apo E ε4 allele is strongly associated with both late-onset familial and sporadic Alzheimer's Disease (AD).

Biallelic markers in linkage disequilibrium with the Apo E ε4 allele were identified. This example is illustrative of the general principle that the generation of biallelic markers associated with a trait leads to markers in linkage disequilibrium with any biallelic marker already known to be associated with the trait.

An Apo E marker was used to screen the human genomic BAC library. A BAC, which gave a unique hybridization signal on chromosomal region 19q13.2.3 by FISH, was selected for finding biallelic markers.

This BAC contained an insert of 205 kb that was subcloned. Fifty BAC subclones were randomly selected and sequenced. Twenty-five subclone sequences were selected and used to design twenty-five couples of PCR primers that allowed amplicons of approximately 500 bp to be generated. These PCR primers were then used to amplify the corresponding genomic sequences in a pool of DNA from 100 individuals (French origin, blood donors) as already described. Amplification products from pooled DNA were sequenced and analyzed for the presence of biallelic polymorphisms using the software described herein. Five amplicons were shown to contain a polymorphic base in the pool of 100 individuals, and therefore these polymorphisms (99-366/274; 99-344/439; 99-365/344; 99-359/308; 99-355/219) were selected as the random biallelic markers in the vicinity of the Apo E gene.

An additional couple of primers was designed that allowed amplification of the genomic fragment carrying the already known polymorphism of Apo E, (99-2452/54 C/T).

An association study was then performed. As expected, there was a clear association between Alzheimer disease (AD) and the known Apo E4 polymorphism (biallelic marker 99-2452/54), the C allele frequency being increased in 26% in the AD case population studied compared to the AD control population analyzed (pvalue of this difference=2×10⁻²¹).

In addition, the association study with the random markers generated in the vicinity of the Apo E gene showed that the biallelic marker 99-365/344 C/T is also associated to AD, the T allele frequency being increased of 17% in the AD case population respect to the AD control population under study (pvalue of this allele frequency difference =7×10⁻¹⁰). Thus individuals who possess a T allele at the biallelic marker 99-365/344 are at risk of developing AD.

Among the biallelic markers generated in the Apo E region, 99-365/344 is in LD with the previously known Apo E4 marker 99-2452/54. The linkage disequilibrium is detected in a control population (LD value=0.08) and is clearly increased in the AD case population (LD=0.21). Hence the generated biallelic marker which are associated with Alzheimer's disease, namely the biallelic marker 99-365, is in linkage disequilibrium with the biallelic marker 99-2452 already known to be associated with this disease.

Identification of a Trait Causing Mutation in the PCTA-1 Gene

If a statistically significant association with a trait is identified for at least one or more of the analyzed PCTA-1-related biallelic markers, one can assume that: either the associated allele is directly responsible for causing the trait, or more likely the associated allele is in linkage disequilibrium with the trait causing allele. More probably, the trait causing mutation would be found near to the associated biallelic markers.

Mutations in the PCTA-1 gene which are responsible for a detectable phenotype may be identified by comparing the sequences of the PCTA-1 gene from trait positive and trait negative individuals. Preferably, trait positive individuals to be sequenced carry a single marker allele or a haplotype shown to be associated to the trait and trait negative individuals to be sequenced do not carry such allele or haplotype associated to the trait. The detectable phenotype may comprise cancer, preferably prostate cancer, a response to or side effects related to a prophylactic or curative agent acting against prostate cancer, the aggressiveness of prostate cancer tumors, expression of the PCTA-1 gene, a modified or forthcoming production of the PCTA-1 protein, or the production of a modified PCTA-1 protein. The mutations may comprise point mutations, deletions, or insertions in the PCTA-1 gene. These mutations are called trait causing mutations and are at least partly responsible for a particular detectable phenotype in an individual. The mutations may lie within the coding sequence for the PCTA-1 protein or within intronic and/or within regulatory regions in the PCTA-1 gene, including splice sites, 5′ UTRs, 3′ UTRs and promoter sequences, including one or more transcription factor binding sites.

A further embodiment of the invention is a method to identify a trait causing mutation in the PCTA-1 gene pursuant to the detection of an association between alleles of one or several of the biallelic markers of the present invention and a particular trait.

This method comprises the following steps:

amplifying a region of the PCTA-1 gene comprising a biallelic marker or a group of biallelic markers associated to the considered trait from DNA samples of trait positive and trait negative individuals;

sequencing the amplified region;

comparing DNA sequences from trait positive and trait negative individuals; and

determining mutations specific to trait positive patients.

In some embodiments, the amplified region is a region located close to a biallelic marker of PCTA-1 gene. In further embodiments, the amplified region is located close to one or more of the biallelic markers A1 to A125 and the complements thereof. In a preferred embodiment, the amplified region is located close to one or more of the biallelic markers A2, A30, A41, AS5, A57 and the complements thereof.

Oligonucleotide primers are constructed as described previously to amplify the sequences of each of the exons, introns, the promoter region and the regulatory regions of the PCTA-1 gene. Amplification is carried out on genomic DNA samples from trait positive patients and trait negative controls, preferably using the PCR conditions described in the examples. Amplification products from the genomic PCRs are then subjected to sequencing, preferably through automated dideoxy terminator sequencing reactions and electrophoresed, preferably on ABI 377 sequencers. Following gel image analysis and DNA sequence extraction, ABI sequence data are automatically analyzed to detect the presence of sequence variations among trait positive and trait negative individuals. Sequences are verified by determining the sequences of both DNA strands for each individual.

Candidate polymorphisms suspected of being responsible for the detectable phenotype, are then verified by screening a larger population of trait positive and trait negative individuals using polymorphism analysis techniques such as the techniques described above. Polymorphisms which exhibit a statistically significant correlation with the detectable phenotype are deemed responsible for the detectable phenotype.

The invention also concerns a mutated PCTA-1 gene comprising a trait causing mutation, and particularly the mutated genes obtained by the process described above.

A mutated PCTA-1 gene can be defined as a gene encoding either a modified or native PCTA-1 protein through a nucleotide sequence which is different from the nucleotide sequence of the PCTA-1 gene found in a majority of trait negative individuals.

The region of the PCTA-1 gene containing the mutation responsible for the detectable phenotype may be used in diagnostic techniques such as those described below. For example, microsequencing oligonucleotides, or oligonucleotides containing the mutation responsible for the detectable phenotype for amplification, or hybridization based diagnostics, such as those described herein, may be used for detecting individuals suffering from the detectable phenotype or individuals at risk of developing the detectable phenotype at a subsequent time. In addition, the PCTA-1 allele responsible for the detectable phenotype may be used in gene therapy. The PCTA-1 allele responsible for the detectable phenotype may also be cloned into an expression vector to express the mutant PCTA-1 protein as described herein.

Biallelic Markers of the Invention in Methods of Genetic Diagnostics

The biallelic markers of the present invention can also be used to develop diagnostics tests capable of identifying individuals who express a detectable trait as the result of a specific genotype or individuals whose genotype places them at risk of developing a detectable trait at a subsequent time. The trait analyzed using the present diagnostics may be any detectable trait, including susceptibility to cancer, preferably prostate cancer, the level of aggressiveness of prostate cancer tumors, an early onset of prostate cancer, a beneficial response to or side effects related to treatment against prostate cancer.

Information resulting from single marker association and for haplotype analyses is extremely valuable as it can, in certain circumstances, be used to initiate preventive treatments or to allow an individual carrying a significant haplotype to foresee warning signs such as minor symptoms. In diseases such as prostate cancer, in which metastasis can be fatal if not stopped in time, the knowledge of a potential predisposition, might contribute in a very significant manner to treatment efficacy. Similarly, a diagnosed predisposition to a potential side-effect could immediately direct the physician toward a treatment for which such side-effects have not been observed during clinical trials.

The invention concerns a method for the detection in an individual of alleles of PCTA-1-related biallelic markers associated with a trait preferably selected from prostate cancer, an early onset of prostate cancer, a susceptibility to prostate cancer, the level of aggressiveness of prostate cancer tumors, or the level of expression of the PCTA-1 gene. The information obtained using this method is useful in the diagnosis, staging, monitoring, prognosis and/or prophylactic or curative therapy of prostate cancer. The method also concerns the detection of specific alleles present within the PCTA-1 gene expressing a modified level of PCTA-1 mRNA or an altered PCTA-1 mRNA, coding for an altered PCTA-1 protein. The identities of the polymorphic bases may be determined using any of the genotyping procedures described above in “Method For Genotyping An Individual For Biallelic Markers”. More particularly, the invention concerns the detection of a PCTA-1 nucleic acid comprising at least one of the nucleotide sequences of P1 to P125 and the complementary sequence thereof. This method comprises the following steps;

obtaining a nucleic acid sample from the individual to be tested; and

determining the presence in the sample of an allele of a biallelic marker or of a group of biallelic markers of the PCTA-1 gene which, when taken alone or in combination with another/other biallelic marker/s of the PCTA-1 gene, is indicative of prostate cancer, of an early onset of prostate cancer, of the level of aggressiveness of prostate cancer tumors, of a modified or forthcoming expression of the PCTA-1 gene, of a modified or forthcoming production of the PCTA-1 protein, or of the production of a modified PCTA-1 protein.

In some embodiments, the biallelic marker comprises at least one of the biallelic markers defined by the sequences P1 to P125, and the complementary sequences thereto, more preferably at least one biallelic marker selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith. In a preferred embodiment, the biallelic marker comprises at least one of the biallelic markers defined by the sequences of P2, P30, P41, P55, P57, and the complementary sequence thereto, more particularly at least one biallelic marker selected from the group consisting of A2, A30, A41, A55, A57 and the complement thereof. In a preferred embodiment, the detection method comprises an additional step of amplifying a nucleotide sequence of the PCTA-1 gene comprising biallelic markers. Optionally, the amplification primers can be selected from the group consisting of B1 to B47 and C1 to C47.

In preferred embodiments of the detection method described above, the presence of alleles of one or more biallelic markers of the PCTA-1 gene is determined through microsequencing reactions. Optionally, the microsequencing primers are selected from the group consisting of D1 to D125 and E1 to E125. Optionally, the microsequencing primers can be bound to a solid support, preferably in the form of arrays of primers attached to appropriate chips or be used in microfluidic devices. Such arrays are described in further detail in the “Oligonucleotide arrays” section. Optionally, the microsequencing primers can be labeled.

In additional preferred embodiments of the detection method, the presence of alleles of one or more biallelic markers of the PCTA-1 gene is determined through an allele specific amplification assay or an enzyme based mismatch detection assay. Optionally, the allele specific amplification assay comprises a step of detecting the presence of the amplification product.

In further preferred embodiments of the detection method, the presence of alleles of one or more biallelic markers of the PCTA-1 gene is determined through a hybridization assay. The probes used in the hybridization assay may include a probe selected from the group consisting of P1 to P125, a complementary sequence thereto or a fragment thereof, said fragment comprising the polymorphic base. Preferably, the probe is labeled.

A diagnostic method according to the present invention can also consist on the detection of an allele of the PCTA-1 gene comprising a trait causing mutation.

The invention also specifically relates to a method of determining whether an individual suffering from prostate cancer or susceptible of developing prostate cancer is likely to respond positively to treatment with a selected medicament acting against prostate cancer.

The method comprises the following steps:

obtaining a DNA sample from the individual to be tested; and

analyzing said DNA sample to determine whether it comprises alleles of one or more biallelic markers associated with a positive response to treatment with the medicament and/or alleles of one or more biallelic markers associated with a negative response to treatment with the medicament.

In a preferred embodiment, the biallelic marker is selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith.

The detection methods of the present invention can be applied to, for example, the preliminary screening of patient populations suffering from prostate cancer. This preliminary screening is useful to initiate adequate treatment when needed or to determine and select appropriate patient populations for clinical trials on new compounds in order to avoid the potential occurrence of specific side effects or to enhance the probability of beneficial patient response. By establishing in advance a homogeneous genotype selection for the population to be tested, the assessment of drug efficacy and/or toxicity can be more readily achieved and less hampered by divergences in population response. This approach can yield better therapeutic approaches based on patient population targeting resulting from pharmacogenomics studies.

The invention also relates to diagnostic kits useful for determining the presence in a DNA sample of alleles associated with the trait, preferably with prostate cancer, with an early onset of prostate cancer, with the level of aggressiveness of prostate cancer tumors, with a modified or forthcoming expression of the PCTA-1 gene, with a modified or forthcoming production of the PCTA-1 protein, or with the production of a modified PCTA-1 protein. Diagnostic kits can comprise any of the polynucleotides of the present invention.

In a first embodiment, the kit comprises primers such as those described above, preferably forward and reverse primers which are used to amplify the PCTA-1 gene or a fragment thereof. In some embodiments, at least one of the primers is complementary to a nucleotide sequence of the PCTA-1 gene comprising a biallelic marker associated with prostate cancer, with an early onset of prostate cancer, with the level of aggressiveness of prostate cancer tumors, with a modified or forthcoming expression of the PCTA-1 gene, with a modified or forthcoming production of the PCTA-1 protein, or with the production of a modified PCTA-1 protein. In one embodiment, the biallelic marker is comprised in one of the sequences P1 to P125 and the complementary sequences thereto. Optionally, the kit comprises an amplification primer which includes a polymorphic base of at least one biallelic marker selected from the group consisting of A1 to A125 and the complements thereof. In a preferred embodiment, the kit comprises one or more of the sequences B1 to B47 and C1 to C47. In a more preferred 1 5 embodiment, the kit comprises one or more of the sequences B1, B16, B20, B23, B24 and C1, C16, C20, C23, C24.

In a second embodiment, the kit comprises microsequencing primers, wherein at least one of said primers is an oligonucleotide capable of hybridizing, either with the coding or with the non-coding strand, immediately upstream of the polymorphic base of a biallelic marker selected from the group consisting of A1 to A125 and the complements thereof, preferably those of D1 to D125 and E1 to E125, more preferably those of D2, D30, D41, D55, D57 and E2, E30, E41, E55, E57.

In a third embodiment, the kit comprises a hybridization DNA probe, that is or eventually becomes immobilized on a solid support, which is capable of hybridizing with the PCTA-1 gene or fragment thereof, preferably which is capable of hybridizing with a region of the PCTA-1 gene which comprises an allele of a biallelic marker of the present invention, more preferably an allele associated with prostate cancer, with an early onset of prostate cancer, with a susceptibility to prostate cancer, with the level of aggressiveness of prostate cancer tumors, with a modified or forthcoming expression of the PCTA-1 gene, with a modified or forthcoming production of the PCTA-1 protein, or with the production of a modified PCTA-1 protein. In a preferred embodiment, the probe is selected from the group consisting of P1 to P125 and the complementary sequences thereto, or a fragment thereof, said fragment comprising the polymorphic base. In a more preferred embodiment, the probe is selected from the group consisting of P2, P30, P41, P55, P57 and the complementary sequences thereto, or a fragment thereof, said fragment comprising the polymorphic base.

The kits of the present invention can also comprise optional elements including appropriate amplification reagents such as DNA polymerases when the kit comprises primers, reagents useful in hybridization reactions and reagents useful to reveal the presence of a hybridization reaction between a labeled hybridization probe and the PCTA-1 gene containing at least one biallelic marker.

Treatment of Cancer or Prostate Cancer

The invention also concerns methods for the treatment of prostate cancer using an allele of a biallelic marker or of a group of biallelic markers, preferably markers of the PCTA-1 gene, associated with a susceptibility to prostate cancer, with an aggressive form of prostate cancer or with a positive or negative response to treatment with an effective amount of a medicament acting against prostate cancer.

As the metastasis of prostate cancer can be fatal, it is important to detect prostate cancer susceptibility of individuals. Consequently, the invention also concerns a method for the treatment of prostate cancer comprising the following steps:

selecting an individual whose DNA comprises alleles of a biallelic marker or of a group of biallelic markers, preferably markers of the PCTA-1 gene, associated with prostate cancer;

following up said individual for the appearance (and optionally the development) of tumors in prostate; and

administering an effective amount of a medicament acting against prostate cancer to said individual at an appropriate stage of the prostate cancer.

In some embodiments, the biallelic marker is comprised in one of the sequences P1 to P125 and the complementary sequences thereto. Preferably the biallelic marker is at least one biallelic marker selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith. In particular embodiments, the individual is selected by genotyping one or more biallelic markers of the present invention.

The prophylactic administration of a treatment serves to prevent, attenuate or inhibit the growth of cancer cells.

Therefore, another embodiment of the present invention consists of a method for the treatment of prostate cancer comprising the following steps:

selecting an individual whose DNA comprises alleles of a biallelic marker or of a group of biallelic markers, preferably markers of the PCTA-1 gene, associated with prostate cancer; and

administering to said individual, preferably as a preventive treatment of prostate cancer, an effective amount of a medicament acting against prostate cancer such as 4HPR or of a vaccine composition capable of conferring immunity against PCTA-1 related prostate cancer.

In some embodiments, the biallelic marker is comprised in one of the sequences P1 to P125 and the complementary sequences thereto. Preferably the biallelic marker is at least one biallelic marker selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith. More preferably the biallelic marker is at least one biallelic marker selected from the group consisting of A2, A30, A41, A55, A57, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith. In particular embodiments, the individual is selected by genotyping one or more biallelic markers of the present invention.

In a further embodiment, the present invention concerns a method for the treatment of prostate cancer comprising the following steps:

selecting an individual whose DNA comprises alleles of a biallelic marker or of a group of biallelic markers, preferably markers of the PCTA-1 gene, associated with a susceptibility prostate cancer;

administering to said individual, as a preventive treatment of prostate cancer, an effective amount of a medicament acting against prostate cancer such as 4HPR or of a vaccine composition capable of conferring immunity against PCTA-1-related prostate cancer;

following up said individual for the appearance and the development of tumors in prostate; and optionally

administering an effective amount of a medicament acting against prostate cancer to said individual at the appropriate stage of the prostate cancer.

In some embodiments, the biallelic marker is comprised in one of the sequences P1 to P125 and the complementary sequences thereto. Preferably the biallelic marker is at least one biallelic marker selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith. More preferably the biallelic marker is at least one biallelic marker selected from the group consisting of A2, A30, A41, A55, A57, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith. In particular embodiments, the individual is selected by genotyping one or more biallelic markers of the present invention.

To enlighten the choice of the appropriate beginning of the treatment of prostate cancer, the present invention also concerns a method for the treatment of prostate cancer comprising the following steps:

selecting an individual suffering from a prostate cancer whose DNA comprises alleles of a biallelic marker or of a group of biallelic markers, preferably markers of the PCTA-1 gene, associated with the aggressiveness of prostate cancer tumors; and

administering an effective amount of a medicament acting against prostate cancer to said individual.

In some embodiments, the biallelic marker is comprised in one of the sequences P1 to P125 and the complementary sequences thereto. Preferably the biallelic marker is at least one biallelic marker selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith. More preferably the biallelic marker is at least one biallelic marker selected from the group consisting of A2, A30, A41, A55, A57, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith. In particular embodiments, the individual is selected by genotyping one or more biallelic markers of the present invention.

The invention concerns a method of determining whether a subject is likely to respond positively to treatment with a selected medicament acting against prostate cancer.

The invention also concerns a method for the treatment of prostate cancer in a selected population of individuals. The method comprises:

selecting an individual suffering from prostate cancer and

whose DNA comprises alleles of a biallelic marker or of a group of biallelic markers, preferably markers of the PCTA-1 gene, associated with a positive response to treatment with an effective amount of a medicament acting against prostate cancer,

and/or whose DNA does not comprise alleles of a biallelic marker or of a group of biallelic markers, preferably markers of the PCTA-1 gene, associated with a negative response to treatment with said medicament; and

administering at suitable intervals an effective amount of said medicament to said selected individual.

In some embodiments, the biallelic marker is comprised in one of the sequences P1 to P125 and the complementary sequences thereto. Preferably the biallelic marker is at least one biallelic marker selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith. In particular embodiments, the individual is selected by genotyping one or more biallelic markers of the present invention.

Another aspect of the invention is a method of using a medicament acting against prostate cancer. The method comprises obtaining a DNA sample from a subject, determining whether the DNA sample contains one or more biallelic markers associated with a positive response to the medicament and/or whether the DNA sample contains one or more biallelic markers associated with a negative response to the medicament, and administering the medicament to the subject if the DNA sample contains one or more biallelic markers associated with a positive response to the medicament and/or if the DNA sample lacks one or more biallelic markers associated with a negative response to the medicament.

The invention also concerns a method for the clinical testing of a medicament, preferably a medicament acting against prostate cancer.

In some embodiments, the medicament may be administered to the subject in a clinical trial if the DNA sample contains alleles of one or more biallelic markers associated with a positive response to treatment with the medicament and/or if the DNA sample lacks alleles of one or more biallelic markers associated with a negative response to treatment with the medicament. In preferred embodiments, the medicament is a drug acting against prostate cancer. In other embodiments, the biallelic marker is selected from the group consisting of A1 to A125 and the complements thereof or optionally the biallelic markers in linkage disequilibrium therewith.

Using the method of the present invention, the evaluation of drug efficacy may be conducted in a population of individuals likely to respond favorably to the medicament.

The invention also concerns a method for the clinical testing of a medicament, preferably a medicament acting against prostate cancer. The method comprises the following steps:

administering a medicament, preferably a medicament susceptible of acting against prostate cancer to a heterogeneous population of individuals;

identifying a first population of individuals who respond positively to said medicament and a second population of individuals who respond negatively to said medicament;

identifying biallelic markers in said first population which are associated with a positive response to said medicament;

selecting individuals whose DNA comprises biallelic markers associated with a positive response to said medicament; and

administering said medicament to said individuals.

Such methods are deemed to be extremely useful to increase the benefit/risk ratio resulting from the administration of medicaments which may cause undesirable side effects and/or be inefficacious to a portion of the patient population to which it is normally administered.

Once an individual has been diagnosed as suffering from a prostate cancer, selection tests are carried out to determine whether the DNA of this individual comprises alleles of a biallelic marker or of a group of biallelic markers associated with a positive response to treatment or with a negative response to treatment which may include either side effects or unresponsiveness.

The selection of the patient to be treated using the method of the present invention can be carried out through the detection methods described above. The individuals which are to be selected are preferably those whose DNA does not comprise alleles of a biallelic marker or of a group of biallelic markers associated with a negative response to treatment. The knowledge of an individual's genetic predisposition to unresponsiveness or side effects to particular medicaments allows the clinician to direct treatment toward appropriate drugs against prostate cancer.

Once the patient's genetic predispositions have been determined, the clinician can select appropriate treatment for which negative response, particularly side effects, has not been reported or has been reported only marginally for the patient.

Recombinant Vectors

The term “vector” is used herein to designate either a circular or a linear DNA or RNA molecule, which is either double-stranded or single-stranded, and which comprise at least one polynucleotide of interest that is sought to be transferred in a cell host or in a unicellular or multicellular host organism.

Another embodiment of the present invention is a recombinant vector. This recombinant vector comprises a nucleotide sequence encoding a regulatory region of the PCTA-1 gene, the promoter region of the PCTA-1 gene, an intron of the PCTA-1 gene, exon 0 and/or exon 1 of the PCTA-1 gene, exon 6bis of the PCTA-1 gene, exon 9bis of the PCTA-1 gene, the genomic sequence of the PCTA-1 gene, a cDNA sequence of the PCTA-1 gene, or combinations of such sequences, or complementary sequences thereto or fragments or variants thereof. Preferred nucleotide sequences included in such an expression vector include at least one nucleotide sequence selected from the group consisting of SEQ ID Nos 1, 2, 3, 4, 8 or fragments or variants thereof or a complementary sequence thereto.

Generally, a recombinant vector of the invention may comprise any of the polynucleotides described herein, including regulatory sequences and coding sequences, as well as any PCTA-1 primer or probe as defined above. More particularly, the recombinant vectors of the present invention can comprise any of the polynucleotides described in the “PCTA-1 cDNA Sequences” section, the “Coding Regions” section, and the “Oligonucleotide Probes And Primers” section.

In another embodiment, the vector includes a PCTA-1 gene or cDNA or a fragment thereof comprising at least one of the biallelic markers described herein, and more preferably a mutated PCTA-1 gene or cDNA comprising a trait causing mutation, particularly a mutation determined using the method described above. Preferably, the biallelic marker is selected from the group consisting of A1 to A125 and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith.

One embodiment of the invention is the production of a PCTA-1 protein under the control of its own promoter or of an exogenous promoter. The present invention also relates to expression vectors which include nucleic acids encoding a native or mutated PCTA-1 protein under the control of either a native PCTA-1 regulatory region, preferably a native PCTA-1 promoter which comprises at least one of the biallelic markers of the present invention, more particularly at least one among the A1 to A43 and the complements thereof, or an exogenous promoter.

More particularly, the present invention relates to expression vectors which include nucleic acids encoding a PCTA-1 protein, preferably a PCTA-1 protein comprising a amino acid sequence selected from the group consisting of SEQ ID Nos 5, 6, 7, 9 or variants or fragments thereof, under the control of a regulatory sequence selected among the PCTA-1 regulatory polynucleotides, or alternatively under the control of an exogenous regulatory sequence.

The present invention also concerns an expression vector comprising a PCTA-1 regulatory region or any sequence thereof of 10 to 3000 nucleotides capable of regulating the expression of a nucleotide sequence encoding a protein and operably linked to the regulatory region. A further preferred regulatory region is the promoter sequence. In this regard, it is to be noted that a portion of the promoter can be used in the expression vector as long as it can influence the transcription of the coding sequence operably linked thereto.

Any nucleotide sequence encoding a polypeptide of interest can be included in an expression vector comprising a PCTA-1 regulatory region and operably linked thereto. Preferred polypeptides are therapeutic proteins which are described in further detail later on.

In some embodiments, expression vectors are employed to express a PCTA-1 polypeptide which can be then purified and, for example be used in ligand screening assays or as an immunogen in order to raise specific antibodies directed against a PCTA-1 protein. In other embodiments, the expression vectors are used for constructing transgenic animals and also for gene therapy.

Some of the elements which can be found in the vectors of the present invention are described in further detail in the following sections.

1. General Features of the Expression Vectors of the Invention

A recombinant vector according to the invention comprises, but is not limited to, a YAC (Yeast Artificial Chromosome), a BAC (Bacterial Artificial Chromosome), a phage, a phagemid, a cosmid, a plasmid or even a linear DNA molecule which may consist of a chromosomal, non-chromosomal, semi-synthetic or synthetic DNA. Such a recombinant vector can comprise a transcriptional unit comprising an assembly of:

(1) a genetic element or elements having a regulatory role in gene expression, for example promoters or enhancers. Enhancers are cis-acting elements of DNA, usually from about 10 to 300 bp in length that act on the promoter to increase the transcription.

(2) a structural or coding sequence which is transcribed into mRNA and eventually translated into a polypeptide, said structural or coding sequence being operably linked to the regulatory elements described in (1); and

(3) appropriate transcription initiation and termination sequences. Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell. Alternatively, when a recombinant protein is expressed without a leader or transport sequence, it may include a N-terminal residue. This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.

Generally, recombinant expression vectors will include origins of replication, selectable markers permitting transformation of the host cell, and a promoter derived from a highly expressed gene to direct transcription of a downstream structural sequence. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably a leader sequence capable of directing secretion of the translated protein into the periplasmic space or the extracellular medium. In a specific embodiment wherein the vector is adapted for transfecting and expressing desired sequences in mammalian host cells, preferred vectors will comprise an origin of replication in the desired host, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′-flanking non-transcribed sequences. DNA sequences derived from the SV40 viral genome, for example SV40 origin, early promoter, enhancer, splice and polyadenylation sites may be used to provide the required non-transcribed genetic elements.

The in vivo expression of a PCTA-1 polypeptide may be useful in order to correct a genetic defect related to the expression of the native gene in a host organism or to the production of a biologically inactive PCTA-1 protein.

Consequently, the present invention also deals with recombinant expression vectors mainly designed for the in vivo production of a PCTA-1 polypeptide of SEQ ID Nos 5, 6, 7, 9 or fragments or variants thereof by the introduction of the appropriate genetic material in the organism of the patient to be treated. This genetic material may be introduced in vitro in a cell that has been previously extracted from the organism, the modified cell being subsequently reintroduced in the said organism, directly in vivo into the appropriate tissue.

2. Regulatory Elements

Promoters

The suitable promoter regions used in the expression vectors according to the present invention are chosen taking into account the cell host in which the heterologous gene has to be I,X;l expressed. The particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of directing the expression of the nucleic acid in the targeted cell. Thus, where a human cell is targeted, it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell, such as, for example, a human or a viral promoter.

A suitable promoter may be heterologous with respect to the nucleic acid for which it controls the expression or alternatively can be endogenous to the native polynucleotide containing the coding sequence to be expressed. Additionally, the promoter is generally heterologous with respect to the recombinant vector sequences within which the construct promoter/coding sequence has been inserted.

Promoter regions can be selected from any desired gene using, for example, CAT (chloramphenicol transferase) vectors and more preferably pKK232-8 and pCM7 vectors.

Preferred bacterial promoters are the LacI, LacZ, the T3 or T7 bacteriophage RNA polymerase promoters, the gpt, lambda PR, PL and trp promoters (EP 0036776, the disclosure of which is incorporated herein by reference in its entirety), the polyhedrin promoter, or the p10 protein promoter from baculovirus (Kit Novagen) (Smith et al., 1983; O'Reilly et al., 1992), the lambda PR promoter or also the trc promoter.

Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionine-L. Selection of a convenient vector and promoter is well within the level of ordinary skill in the art.

The choice of a promoter is well within the ability of a person skilled in the field of genetic engineering. For example, one may refer to the book of Sambrook et al.(1989) or also to the procedures described by Fuller et al.(1996).

Other Regulatory Elements

Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed such as human growth hormone and SV40 polyadenylation signals. Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.

The vector containing the appropriate DNA sequence as described above, more preferably PCTA-1 gene regulatory polynucleotide, a polynucleotide encoding a PCTA-1 polypeptide selected from the group consisting of SEQ ID No 1 or a fragment or a variant thereof and SEQ ID Nos 2, 3,4, 8, or both of them, can be utilized to transform an appropriate host to allow the expression of the desired polypeptide or polynucleotide.

3. Selectable Markers

Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct. The selectable marker genes for selection of transformed host cells are preferably dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, TRPI for S. cerevisiae or tetracycline, rifampicin or ampicillin resistance in E. coli, or levan saccharase for mycobacteria, this latter marker being a negative selection marker.

4. Preferred Vectors.

Bacterial Vectors

As a representative but non-limiting example, useful expression vectors for bacterial use can comprise a selectable marker and a bacterial origin of replication derived from commercially available plasmids comprising genetic elements of pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia, Uppsala, Sweden), and GEMI (Promega Biotec, Madison, Wis., USA).

Large numbers of other suitable vectors are known to those of skill in the art, and commercially available, such as the following bacterial vectors: pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia); pQE-30 (QIAexpress).

Bacteriophage Vectors

The P1 bacteriophage vector may contain large inserts ranging from about 80 to about 100 kb.

The construction of P1 bacteriophage vectors such as p158 or p158/neo8 are notably described by Sternberg (1994). Recombinant P1 clones comprising PCTA-1 nucleotide sequences may be designed for inserting large polynucleotides of more than 40 kb (Linton et al., 1993). To generate P1 DNA for transgenic experiments, a preferred protocol is the protocol described by McCormick et al.(1994). Briefly, E. coli (preferably strain NS3529) harboring the P1 plasmid are grown overnight in a suitable broth medium containing 25 μg/ml of kanamycin. The P1 DNA is prepared from the E. coli by alkaline lysis using the Qiagen Plasmid Maxi kit (Qiagen, Chatsworth, Calif., USA), according to the manufacturer's instructions. The P1 DNA is purified from the bacterial lysate on two Qiagen-tip 500 columns, using the washing and elution buffers contained in the kit. A phenol/chloroform extraction is then performed before precipitating the DNA with 70% ethanol. After solubilizing the DNA in TE (10 mM Tris-HCl, pH 7.4, 1 mM EDTA), the concentration of the DNA is assessed by spectrophotometry.

When the goal is to express a P1 clone comprising PCTA-1 nucleotide sequences in a transgenic animal, typically in transgenic mice, it is desirable to remove vector sequences from the P1 DNA fragment, for example by cleaving the P1 DNA at rare-cutting sites within the P1 polylinker (SfiI, NotI or SalI). The P1 insert is then purified from vector sequences on a pulsed-field agarose gel, using methods similar using methods similar to those originally reported for the isolation of DNA from YACs (Schedl et al., 1993a; Peterson et al., 1993). At this stage, the resulting purified insert DNA can be concentrated, if necessary, on a Millipore Ultrafree-MC Filter Unit (Millipore, Bedford, Mass., USA-30,000 molecular weight limit) and then dialyzed against microinjection buffer(10 mM Tris-HCl, pH 7.4; 250 μM EDTA) containing 100 mM NaCl, 30 μM spermine, 70 μM spermidine on a microdyalisis membrane (type VS, 0.025 μM from Millipore). The intactness of the purified P1 DNA insert is assessed by electrophoresis on 1% agarose (Sea Kem GTG; FMC Bio-products) pulse-field gel and staining with ethidium bromide.

Baculovirus Vectors

A suitable vector for the expression of a PCTA-1 polypeptide of SEQ ID Nos 5, 6, 7, 9 or fragments or variants thereof is a baculovirus vector that can be propagated in insect cells and in insect cell lines. A specific suitable host vector system is the pVL1392/1393 baculovirus transfer vector (Pharmingen) that is used to transfect the SF9 cell line (ATCC N^(o)CRL 1711) which is derived from Spodoptera frugiperda.

Other suitable vectors for the expression of a PCTA-1 polypeptide of SEQ ID Nos 5, 6, 7, 9 or fragments or variants thereof in a baculovirus expression system include those described by Chai et al.(l993), Vlasak et al.(l983) and Lenhard et al.(1996).

Viral Vectors

In one specific embodiment, the vector is derived from an adenovirus. Preferred adenovirus vectors according to the invention are those described by Feldman and Steg (1996) or Ohno et al.(l994). Another preferred recombinant adenovirus according to this specific embodiment of the present invention is the human adenovirus type 2 or 5 (Ad 2 or Ad 5) or an adenovirus of animal origin (French patent application N^(o)FR-93.05954).

Retrovirus vectors and adeno-associatcd virus vectors are generally understood to be the recombinant gene delivery systems of choice for the transfer of exogenous polynucleotides in vivo, particularly to mammals, including humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal. DNA of the host.

Particularly preferred retroviruses for the preparation or construction of retroviral in vitro or in vitro gene delivery vehicles of the present invention include retroviruses selected from the group consisting of Mink-Cell Focus Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis virus and Rous Sarcoma virus. Particularly preferred Murine Leukemia Viruses include the 4070A and the 1504A viruses, Abelson (ATCC No VR-999), Friend (ATCC No VR-245), Gross (ATCC No VR-590), Rauscher (ATCC No VR-998) and Moloney Murine Leukemia Virus (ATCC No VR-190; PCT Application No WO 94/24298). Particularly preferred Rous Sarcoma Viruses include Bryan high titer (ATCC Nos VR-334, VR-657, VR-726, VR-659 and VR-728). Other preferred retroviral vectors are those described in Roth et al.(1996), PCT Application No WO 93/25234 (the disclosure of which is incorporated herein by reference in its entirety), PCT Application No WO 94/ 06920 (the disclosure of which is incorporated herein by reference in its entirety), Roux et al., 1989, Julan et al., 1992 and Neda et al., 1991.

Yet another viral vector system that is contemplated by the invention consists of the adeno-associated virus (AAV). The adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle (Muzyczka et al., 1992). It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (Flotte et al., 1992; Samulski et al., 1989; McLaughlin et al., 1989). One advantageous feature of AAV derives from its reduced efficacy for transducing primary cells relative to transformed cells.

BAC Vectors

The bacterial artificial chromosome (BAC) cloning system (Shizuya et al , 1992) has been developed to stably maintain large fragments of genomic DNA (100-300 kb) in E. coli. A preferred BAC vector consists of pBeloBAC 11 vector that has been described by Kim et al.(1996). BAC libraries are prepared with this vector using size-selected genomic DNA that has been partially digested using enzymes that permit ligation into either the Bam HI or HindIII sites in the vector. Flanking these cloning sites are T7 and SP6 RNA polymerase transcription initiation sites that can be used to generate end probes by either RNA transcription or PCR methods. After the construction of a BAC library in E. coli, BAC DNA is purified from the host cell as a supercoiled circle. Converting these circular molecules into a linear form precedes both size determination and introduction of the BACs into recipient cells. The cloning site is flanked by two Not I sites, permitting cloned segments to be excised from the vector by Not I digestion. Alternatively, the DNA insert contained in the pBeloBAC11 vector may be linearized by treatment of the BAC vector with the commercially available enzyme lambda terminase that leads to the cleavage at the unique cosN site, but this cleavage method results in a full length BAC clone containing both the insert DNA and the BAC sequences.

5. Delivery of the Recombinant Vectors

In order to effect expression of the polynucleotides and polynucleotide constructs of the invention, these constructs must be delivered into a cell. This delivery may be accomplished in vitro, as in laboratory procedures for transforming cell lines, or in vivo or ex vivo, as in the treatment of certain diseases states.

One mechanism is viral infection where the expression construct is encapsulated in an infectious viral particle.

Several non-viral methods for the transfer of polynucleotides into cultured mammalian cells are also contemplated by the present invention, and include, without being limited to, calcium phosphate precipitation (Graham et al., 1973; Chen et al., 1987;), DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984), direct microinjection (Harland et al., 1985), DNA-loaded liposomes (Nicolau et al., 1982; Fraley et al., 1979), and receptor-mediate transfection (Wu and Wu, 1987; 1988). Some of these techniques may be successfully adapted for in vivo or ex vivo use.

Once the expression polynucleotide has been delivered into the cell, it may be stably integrated into the genome of the recipient cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non specific location (gene augmentation). In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or “episomes” encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle.

One specific embodiment for a method for delivering a protein or peptide to the interior of a cell of a vertebrate in vivo comprises the step of introducing a preparation comprising a physiologically acceptable carrier and a naked polynucleotide operatively coding for the polypeptide of interest into the interstitial space of a tissue comprising the cell, whereby the naked polynucleotide is taken up into the interior of the cell and has a physiological effect. This is particularly applicable for transfer in vitro but it may be applied to in vivo as well.

Compositions for use in vitro and in vivo comprising a “naked” polynucleotide are described in PCT application N^(o) WO 90/11092 (Vical Inc.) and also in PCT application No. WO 95/11307 (Institut Pasteur, INSERM, Universitéd'Ottawa) as well as in the articles of Tacson et al.(l996) and of Huygen et al.(1996).

In still another embodiment of the invention, the transfer of a naked polynucleotide of the invention, including a polynucleotide construct of the invention, into cells may be proceeded with a particle bombardment (biolistic), said particles being DNA-coated microprojectiles accelerated to a high velocity allowing them to pierce cell membranes and enter cells without killing them, such as described by Klein et al.(1987).

In a further embodiment, the polynucleotide of the invention may be entrapped in a liposome (Ghosh and Bacchawat, 1991; Wong et al., 1980; Nicolau et al., 1987)

In a specific embodiment, the invention provides a composition for the in vivo production of a PCTA-1 protein or polypeptide described herein. It comprises a naked polynucleotide operatively coding for this polypeptide, in solution in a physiologically acceptable carrier, and suitable for introduction into a tissue to cause cells of the tissue to express the said protein or polypeptide.

The amount of vector to be injected to the desired host organism varies according to the site of injection. As an indicative dose, it will be injected between 0,1 and 100 μg of the vector in an animal body, preferably a mammal body, for example a mouse body.

In another embodiment of the vector according to the invention, it may be introduced in vitro in a host cell, preferably in a host cell previously harvested from the animal to be treated and more preferably a somatic cell such as a muscle cell. In a subsequent step, the cell that has been transformed with the vector coding for the desired PCTA-1 polypeptide or the desired fragment thereof is reintroduced into the animal body in order to deliver the recombinant protein within the body either locally or systemically.

Cell Hosts

The invention also concerns host cells transformed by one of the vectors described above that produce either a heterologous protein, a PCTA-1 protein or fragments thereof encoded by the PCTA-1 gene, preferably comprising at least one of the biallelic polymorphisms described herein, and more preferably a mutated PCTA-1 gene comprising the trait causing mutation determined using the above-noted method.

Another object of the invention consists of a host cell that has been transformed or transfected with one of the polynucleotides described herein, and in particular a polynucleotide either comprising a PCTA-1 regulatory polynucleotide or the coding sequence of a PCTA-1 polypeptide selected from the group consisting of SEQ ID No 1 2, 3, 4, 8 or a fragment or a variant thereof. Also included are host cells that are transformed (prokaryotic cells) or that are transfected (eukaryotic cells) with a recombinant vector such as one of those described above. More particularly, the cell hosts of the present invention can comprise any of the polynucleotides described in the “PCTA-1 cDNA Sequences” section, the “Coding Regions” section, and the “Oligonucleotide Probes And Primers” section.

A further recombinant cell host according to the invention comprises a polynucleotide containing a biallelic marker selected from the group consisting of A1 to A125, and the complements thereof.

Generally, a recombinant host cell of the invention comprises any one of the polynucleotides or the recombinant vectors described herein.

Preferred host cells used as recipients for the expression vectors of the invention are the following:

a) Prokaryotic host cells: Escherichia coli strains (I.E.DH5-α strain), Bacillus subtilis, Salmonella typhimurium, and strains from species like Pseudomonas, Streptomyces and Staphylococcus.

b) Eukaryotic host cells: HeLa cells (ATCC N^(o)CCL2; N^(o)CCL2. 1; N^(o)CCL2.2), Cv 1 cells (ATCC N^(o)CCL70), COS cells (ATCC N^(o)CRL1650; N^(o)CRL1651), Sf-9 cells (ATCC N^(o)CRL1711), C127 cells (ATCC N^(o)CRL-1804),3T3 (ATCC N^(o)CRL-6361), CHO (ATCC N^(o)CCL-61), human kidney 293. (ATCC N^(o) 45504; N^(o)CRL-1573) and BHK (ECACC N^(o)84100501; N^(o) 84111301).

c) Other mammalian host cells.

The PCTA-1 gene expression in mammalian, and typically human, cells may be rendered defective, or alternatively it may be proceeded with the insertion of a PCTA-1 genomic or cDNA sequence with the replacement of the PCTA-1 gene counterpart in the genome of an animal cell by a PCTA-1 polynucleotide according to the invention. These genetic alterations may be generated by homologous recombination events using specific DNA constructs that have been previously described.

One kind of cell hosts that may be used are mammal zygotes, such as murine zygotes. For example, murine zygotes may undergo microinjection with a purified DNA molecule of interest, for example a purified DNA molecule that has previously been adjusted to a concentration range from 1 ng/ml -for BAC inserts- 3 ng/μl -for P1 bacteriophage inserts- in 10 mM Tris-HCl, pH 7.4, 250 μM EDTA containing 100 mM NaCl, 30 μM spermine, and70 μM spermidine. When the DNA to be microinjected has a large size, polyamines and high salt concentrations can be used in order to avoid mechanical breakage of this DNA, as described by Schedl et al (1993b).

Anyone of the polynucleotides of the invention, including the DNA constructs described herein, may be introduced in an embryonic stem (ES) cell line, preferably a mouse ES cell line. ES cell lines are derived from pluripotent, uncommitted cells of the inner cell mass of pre-implantation blastocysts. Preferred ES cell lines are the following: ES-E 14TG2a (ATCC n^(o) CRL-1821), ES-D3 (ATCC n^(o) CRL1934 and n^(o) CRL-11632), YS001 (ATCC n^(o) CRL-11776),36.5 (ATCC n^(o) CRL-11116). To maintain ES cells in an uncommitted state, they are cultured in the presence of growth inhibited feeder cells which provide the appropriate signals to preserve this embryonic phenotype and serve as a matrix for ES cell adherence. Preferred feeder cells consist of primary embryonic fibroblasts that are established from tissue of day 13-day 14 embryos of virtually any mouse strain, that are maintained in culture, such as described by Abbondanzo et al.(1993) and are inhibited in growth by irradiation, such as described by Robertson (1987), or by the presence of an inhibitory concentration of LIF, such as described by Pease and Williams (1990).

The constructs in the host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.

Following transformation of a suitable host and growth of the host to an appropriate cell density, the selected promoter is induced by appropriate means, such as temperature shift or chemical induction, and cells are cultivated for an additional period.

Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

Microbial cells employed in the expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. Such methods are well known by the skill artisan.

Transgenic Animals

The invention also relates to transgenic animals having an exogenous PCTA-1 regulatory region or a PCTA-1 gene, preferably comprising at least one of the biallelic polymorphisms described herein, and more preferably to a mutated PCTA-1 gene comprising the trait causing mutation determined using the above-noted method. Preferably, the biallelic marker is selected from the group consisting of A1 to A125 and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith. In another embodiment, the invention concerns animals, preferably a mouse, having the mouse PCTA-1 gene which is modified or knocked out. These animals could be used to screen compounds of interest.

The terms “transgenic animals” or “host animals” are used herein to designate animals that have their genome genetically and artificially manipulated so as to include one of the nucleic acids according to the invention. Preferred animals are non-human mammals and include those belonging to a genus selected from Mus (e.g. mice), Rattus (e.g. rats) and Oryctogalus (e.g. rabbits) which have their genome artificially and genetically altered by the insertion of a nucleic acid according to the invention.

In one embodiment, the invention encompasses non-human host mammals and animals comprising a recombinant vector of the invention, a polynucleotide construct according to the invention, or a PCTA-1 gene disrupted by homologous recombination with a knock out vector. Generally, a transgenic animal according the present invention comprises any one of the polynucleotides, the recombinant vectors and the cell hosts described in the present invention. More particularly, the transgenic animals according to the present invention can comprise any of the polynucleotides described in the “PCTA-1 cDNA Sequences” section, the “Coding Regions” section, and the “Oligonucleotide Probes And Primers” section.

The transgenic animals of the invention all include within a plurality of their cells a cloned recombinant or synthetic DNA sequence, more specifically one of the purified or isolated nucleic acids comprising a PCTA-1 coding sequence, a PCTA-1 regulatory polynucleotide or a DNA sequence encoding an antisense polynucleotide such as described in the present specification, and still more preferably a nucleotide comprising an allele of at least one biallelic marker of the PCTA-1 gene.

In a first preferred embodiment, these transgenic animals may be good experimental models in order to study cancer, preferably prostate cancer, in particular concerning the transgenic animals within the genome of which has been inserted one or several copies of a polynucleotide encoding a native PCTA-1 protein, or alternatively a mutant PCTA-1 protein.

In a second preferred embodiment, these transgenic animals may express a desired polypeptide of interest under the control of the regulatory polynucleotides of the PCTA-1 gene, leading to good yields in the synthesis of this protein of interest, and eventually a tissue specific expression of this protein of interest.

The design of the transgenic animals of the invention may be made according to the conventional techniques well known from the one skilled in the art. For more details regarding the production of transgenic animals, and specifically transgenic mice, it may be referred to U.S. Pat. Nos. 4,873,191, issued Oct. 10, 1989; U.S. Pat. No. 5,464,764 issued Nov. 7, 1995; and U.S. Pat. No. 5,789,215, issued Aug. 4, 1998, the disclosures of which are incorporated herein by reference in their entireties.

Transgenic animals of the present invention are produced by the application of procedures which result in an animal with a genome that incorporates exogenous genetic material which is integrated into the genome. The procedure involves obtaining the genetic material, or a portion thereof, which encodes either a PCTA-1 coding sequence, a PCTA-1 regulatory polynucleotide or a DNA sequence encoding an antisense polynucleotide such as described in the present specification.

A recombinant polynucleotide of the invention is inserted into an embryonic or ES stem cell line. The insertion is made using electroporation. The cells subjected to electroporation are screened (e.g. Southern blot analysis) to find positive cells which have integrated the exogenous recombinant polynucleotide into their genome. An illustrative positive-negative selection procedure that may be used according to the invention is described by Mansour et al. (1988).

Then, the positive cells are isolated, cloned and injected into 3.5 days old blastocysts from mice. The blastocysts are then inserted into a female host animal and allowed to grow to term.

Alternatively, the positive ES cells are brought into contact with embryos at the 2.5 days old 8-16 cell stage (morulae) such as described by Wood et al.(1993) or by Nagy et al.(1993), the ES cells being intemalized to colonize extensively the blastocyst including the cells which will give rise to the germ line.

The offsprings of the female host are tested to determine which animals are transgenic e.g. include the inserted exogenous DNA sequence and which are wild-type.

Thus, the present invention also concerns a transgenic animal containing a nucleic acid, a recombinant expression vector or a recombinant host cell according to the invention.

Recombinant Cell Lines Derived from the Transgenic Animals of the Invention.

A further object of the invention consists of recombinant host cells obtained from a transgenic animal described herein. In one embodiment the invention encompasses cells derived from non-human host mammals and animals comprising a recombinant vector of the invention or a PCTA-1 gene disrupted by homologous recombination with a knock out vector.

Recombinant cell lines may be established in vitro from cells obtained from any tissue of a transgenic animal according to the invention, for example by transfection of primary cell cultures with vectors expressing one-genes such as SV40 large T antigen, as described by Chou (1989) and Shay et al.(l991).

Screening of Agents Acting Against Prostate Cancer

In a further embodiment, the present invention also concerns a method for the screening of new agents, or candidate substances, acting against cancer, preferably against prostate cancer and which may be suitable for the treatment of a patient whose DNA comprises an allele of the PCTA-1 gene associated with cancer, preferably with prostate cancer, with an early onset of prostate cancer, or with the aggressiveness of prostate cancer tumors, or more generally with a modified or forthcoming expression of the PCTA-1 gene, with a modified or forthcoming production of the PCTA-1 protein, or with the production of a modified PCTA-1 protein.

In a preferred embodiment, the invention relates to a method for the screening of candidate substances for cancer treatment, preferably prostate cancer treatment. The method comprises the following steps:

providing a cell line, an organ, or a mammal expressing a PCTA-1 gene or a fragment thereof, preferably the regulatory region or the promoter region of the PCTA-1 gene;

obtaining a candidate substance, preferably a candidate substance capable of inhibiting the binding of a transcription factor to the PCTA-1 regulatory region; and

testing the ability of the candidate substance to decrease the symptoms of cancer, preferably of prostate cancer and/or to modulate the expression levels of PCTA-1.

In some embodiments, the cell line, organ or mammal expresses a heterologous protein, the coding sequence of which is operably linked to the PCTA-1 regulatory or promoter sequence. In other embodiments, they express a PCTA-1 gene comprising alleles of one or more biallelic markers associated with cancer, preferably with prostate cancer, an early onset of prostate cancer, or the aggressiveness of prostate cancer tumors, or a mutated PCTA-1 gene comprising a trait causing mutation determined using the above-noted method. Optionally, the biallelic marker is selected from the group consisting of A1 to A125 and the complements thereof. Preferably, the biallelic marker is selected from the group consisting of A2, A30, A41, A55, A57 and the complements thereof. In a further embodiment, a mice expressing a PCTA-1 protein, preferably a mouse PCTA-1 protein encoded by a nucleic acid sequence of SEQ ID No 9 or a variant or a fragment thereof can be used to screen agents acting against cancer, preferably prostate cancer.

A candidate substance is a substance which can interact with or modulate, by binding or other intermolecular interactions, expression, stability, and function of PCTA-1. Such substances may be potentially interesting for patients who are not responsive to existing drugs or develop side effects to them. Screening may be effected using either in vitro methods or in vivo methods.

Such methods can be carried out in numerous ways such as on transformed cells which express the considered alleles of the PCTA-1 gene, on tumors induced by said transformed cells, for example in mice, or on PCTA-1 protein encoded by the considered allelic variant of PCTA-1. This method preferably includes preparing transformed cells with different forms of PCTA-1 sequences containing particular alleles of one or more biallelic markers and/or trait causing mutations described above. Optionally, the biallelic marker is selected from the group consisting of A1 to A125 and the complements thereof.

Screening assays of the present invention generally involve determining the ability of a candidate substance to present a cytotoxic effect, to change the characteristics of transformed cells such as proliferative and invasive capacity, to affect the tumor growth, or to modify the expression level of PTCA-1.

Typical examples of such drug screening assays are provided below. It is to be understood that the parameters set forth in these examples can be modified by the skilled person without undue experimentation.

Screening Substances Interacting with the Regulatory Sequences of the PCTA-1 Gene.

The present invention also concerns a method for screening substances or molecules that are able to interact with the regulatory sequences of the PCTA-1 gene, such as for example promoter or enhancer sequences.

Nucleic acids encoding proteins which are able to interact with the regulatory sequences of the PCTA-1 gene, more particularly a nucleotide sequence selected from the group consisting of the polynucleotides of the 5′ and 3′ regulatory region or a fragment or variant thereof, and preferably a variant comprising one of the biallelic markers of the invention, may be identified by using a one-hybrid system, such as that described in the booklet enclosed in the Matchmaker One-Hybrid System kit from Clontech (Catalog Ref. n^(o) K1603-1). Briefly, the target nucleotide sequence is cloned upstream of a selectable reporter sequence and the resulting DNA construct is integrated in the yeast genome (Saccharomyces cerevisiae). The yeast cells containing the reporter sequence in their genome are then transformed with a library consisting of fusion molecules between cDNAs encoding candidate proteins for binding onto the regulatory sequences of the PCTA-1 gene and sequences encoding the activator domain of a yeast transcription factor such as GAL4. The recombinant yeast cells are plated in a culture broth for selecting cells expressing the reporter sequence. The recombinant yeast cells thus selected contain a fusion protein that is able to bind onto the target regulatory sequence of the PCTA-1 gene. Then, the cDNAs encoding the fusion proteins are sequenced and may be cloned into expression or transcription vectors in vitro. The binding of the encoded polypeptides to the target regulatory sequences of the PCTA-1 gene may be confirmed by techniques familiar to the one skilled in the art, such as gel retardation assays or DNAse protection assays. Such assays are detailed in the section “Analysis Of Biallelic Markers Of The Invention With Prostate Cancer”.

Screening for Expression Modifiers

The PCTA-1 gene appears to be involved in a series of events which most likely include a modification of at least one step of its transcription process. In fact, and as mentioned previously, there is a strong possibility that this modification is directly related to the binding efficiency of DNA binding factors to sites of the PCTA-1 regulatory region.

Screening programs can be used to test potentially therapeutic compounds, either by competitively binding to the sites of the PCTA-1 promoter which would normally bind the DNA transcription factor, or directly binding to the DNA binding factor itself. These compounds could reduce the speed at which the cascade of events leading to the development of PCTA-1 related cancers takes place. In fact, even though it seems clear that a combination of several DNA binding sites may be involved in the development of a PCTA-1 related prostate cancer, binding inhibition of only a few such sites is likely to be sufficient to significantly impact on PCTA-1 production and hence the proliferation of cancer.

The screening of expression modifiers is important as it can be used for detecting modifiers specific to one allele or a group of alleles of the PCTA-1 gene. The alteration of PCTA-1 expression in response to a modifier can be determined by administering or combining the candidate modifier with an expression system such as animals, cells, and in vitro transcription assay.

The term “expression modifier” is intended to encompass but is not limited to chemical agents and polypeptides that modulate the action of PCTA-1 through modulation of the PCTA-1 gene expression.

The effect of the modifier on PCTA-1 transcription and/or steady state mRNA levels can be also determined. As with the basic expression levels, tissue specific interactions are of interest. Correlations are made between the ability of an expression modifier to affect PCTA-1 activity, and the presence of the targeted polymorphisms. A panel of different modifiers may be screened in order to determine the effect under a number of different conditions.

Another subject of the present invention is a method for screening molecules that modulate the expression of the PCTA-1 protein. Such a screening method comprises the steps of:

a) cultivating a prokaryotic or an eukaryotic cell that has been transfected with a nucleotide sequence encoding the PCTA-1 protein or a variant or a fragment thereof, placed under the control of its own promoter;

b) bringing into contact the cultivated cell with a molecule to be tested; and

c) quantifying the expression of the PCTA-1 protein or a variant or a fragment thereof.

In an embodiment, the nucleotide sequence encoding the PCTA-1 protein or a variant or a fragment thereof comprises an allele of at least one of the biallelic markers A1 to A125, preferably A2, A30, A41, A55, A57, and the complements thereof.

Using DNA recombination techniques well known by the one skilled in the art, the PCTA-1 protein encoding DNA sequence is inserted into an expression vector, downstream from its promoter sequence.

The quantification of the expression of the PCTA-1 protein may be realized either at the mRNA level or at the protein level. In the latter case, polyclonal or monoclonal antibodies may be used to quantify the amounts of the PCTA-1 protein that have been produced, for example in an ELISA or a RIA assay.

In a preferred embodiment, the quantification of the PCTA-1 mRNA is realized by a quantitative PCR amplification of the cDNA obtained by a reverse transcription of the total mRNA of the cultivated PCTA-1-transfected host cell, using a pair of primers specific for PCTA-1.

Thus, is also part of the present invention a method for screening of a candidate substance or molecule that modulated the expression of the PCTA-1 gene, this method comprises the following steps:

providing a recombinant cell host containing a nucleic acid, wherein said nucleic acid comprises a nucleotide sequence of the 5′ regulatory region or a biologically active fragment or variant thereof located upstream a polynucleotide encoding a detectable protein;

obtaining a candidate substance; and

determining the ability of the candidate substance to modulate the expression levels of the polynucleotide encoding the detectable protein.

In a further embodiment, the nucleic acid comprising the nucleotide sequence of the 5′ regulatory region or a biologically active fragment or variant thereof also includes a 5′UTR region of the PCTA-1 cDNAs, or one of its biologically active fragments or variants thereof.

Among the preferred polynucleotides encoding a detectable protein, there may be cited polynucleotides encoding luciferase, beta galactosidase, green fluorescent protein (GFP) and chloramphenicol acetyl transferase (CAT).

In another embodiment of a method for the screening of a candidate substance or molecule that modulates the expression of the PCTA-1 gene, wherein said method comprises the following steps:

a) providing a recombinant host cell containing a nucleic acid, wherein said nucleic acid comprises the 5′UTR sequence of a PCTA-1 cDNA, or one of its biologically active fragments or variants, the 5′UTR sequence or its biologically active fragment or variant being operably linked to a polynucleotide encoding a detectable protein;

b) obtaining a candidate substance; and

c) determining the ability of the candidate substance to modulate the expression levels of the polynucleotide encoding the detectable protein.

In one particular embodiment of the above screening method, the nucleic acid that comprises a nucleotide sequence selected from the group consisting of the 5′UTR sequence of a PCTA-1 cDNA or one of its biologically active fragments or variants, includes a promoter sequence which is exogenous with respect to the PCTA-1 5′UTR sequence defined therein. In a further preferred embodiment, the nucleic acid comprising the 5′-UTR sequence of a PCTA-1 cDNA or the biologically active fragments thereof includes a biallelic marker selected from the group consisting of A1 to A125, preferably A2, A30, A41, A55, A57, or the complements thereof.

The invention also pertains to kits useful for performing the herein described screening method. Preferably, such kits comprise a recombinant vector that allows the expression of a nucleotide sequence of the 5′ regulatory region or a biologically active fragment or variant thereof located upstream and operably linked to a polynucleotide encoding a detectable protein or a PCTA-1 protein or a fragment or a variant thereof. Moreover, the kit can comprise a recombinant vector that comprises a nucleic acid including a 5′UTR sequence of a PCTA-1 cDNA, or one of their biologically active fragments or variants, said nucleic acid being operably linked to a polynucleotide encoding a detectable protein or a PCTA-1 protein or a fragment or a variant thereof.

For the design of suitable recombinant vectors useful for performing the screening methods described above, it will be referred to the section of the present specification wherein the preferred recombinant vectors of the invention are detailed.

Expression levels and patterns of PCTA-1 may be analyzed by solution hybridization with long probes as described in International Patent Application No. WO 97/05277. Briefly, a PCTA-1 cDNA or the PCTA-1 genomic DNA described above, or fragments thereof, is inserted at a cloning site immediately downstream of a bacteriophage (T3, T7 or SP6) RNA polymerase promoter to produce antisense RNA. Preferably, the PCTA-1 insert comprises at least 100 or more consecutive nucleotides of the genomic DNA sequence or a cDNA sequence, particularly those comprising at least one of biallelic markers according the present invention, preferably at least one of the biallelic markers A1 to A125 and the complements thereof or those comprising the trait causing mutation. The plasmid is linearized and transcribed in the presence of ribonucleotides comprising modified ribonucleotides (i.e. biotin-UTP and DIG-UTP). An excess of this doubly labeled RNA is hybridized in solution with mRNA isolated from cells or tissues of interest. The hybridizations are performed under standard stringent conditions (40-50° C. for 16 hours in an 80% formamide, 0.4 M NaCl buffer, pH 7-8). The unhybridized probe is removed by digestion with ribonucleases specific for single-stranded RNA (i.e. RNases CL3, T1, Phy M, U2 or A). The presence of the biotin-UTP modification enables capture of the hybrid on a microtitration plate coated with streptavidin. The presence of the DIG modification enables the hybrid to be detected and quantified by ELISA using an anti-DIG antibody coupled to alkaline phosphatase.

Quantitative analysis of the PCTA-1 gene expression may also be performed using arrays. As used herein, the term array means a one dimensional, two dimensional, or multidimensional arrangement of a plurality of nucleic acids of sufficient length to permit specific detection of expression of mRNAs capable of hybridizing thereto. For example, the arrays may contain a plurality of nucleic acids derived from genes whose expression levels are to be assessed. The arrays may include the PCTA-1 genomic DNA, a PCTA-1 cDNA sequence or the sequences complementary thereto or fragments thereof, particularly those comprising at least one of the biallelic markers according the present invention, preferably at least one of the biallelic markers A1 to A125 and the complements thereof or those comprising a trait causing mutation. Preferably, the fragments are at least 15 nucleotides in length. In other embodiments, the fragments are at least 25 nucleotides in length. In some embodiments, the fragments are at least 50 nucleotides in length. More preferably, the fragments are at least 100 nucleotides in length. In another preferred embodiment, the fragments are more than 100 nucleotides in length. In some embodiments the fragments may be more than 500 nucleotides in length.

For example, quantitative analysis of PCTA-1 gene expression may be performed with a complementary DNA microarray as described by Schena et al. (1995 and 1996). Full length PCTA-1 cDNAs or fragments thereof are amplified by PCR and arrayed from a 96-well microtiter plate onto silylated microscope slides using high-speed robotics. Printed arrays are incubated in a humid chamber to allow rehydration of the array elements and rinsed, once in 0.2% SDS for 1 min, twice in water for 1 min and once for 5 min in sodium borohydride solution. The arrays are submerged in water for 2 min at 95° C., transferred into 0.2% SDS for 1 min, rinsed twice with water, air dried and stored in the dark at 25° C.

Cell or tissue mRNA is isolated or commercially obtained and probes are prepared by a single round of reverse transcription. Probes are hybridized to 1 cm² microarrays under a 14×14 mm glass coverslip for 6-12 hours at 60° C. Arrays are washed for 5 min at 25° C. in low stringency wash buffer (1×SSC/0.2% SDS), then for 10 min at room temperature in high stringency wash buffer (0.1×SSC/0.2% SDS). Arrays are scanned in 0.1×SSC using a fluorescence laser scanning device fitted with a custom filter set. Accurate differential expression measurements are obtained by taking the average of the ratios of two independent hybridizations.

Quantitative analysis of PCTA-1 gene expression may also be performed with full length PCTA-1 cDNAs or fragments thereof in complementary DNA arrays as described by Pietu et al. (1996). The full length PCTA-1 cDNA or fragments thereof is PCR amplified and spotted on membranes. Then, mRNAs originating from various tissues or cells are labeled with radioactive nucleotides. After hybridization and washing in controlled conditions, the hybridized mRNAs arc detected by phospho-imaging or autoradiography. Duplicate experiments are performed and a quantitative analysis of differentially expressed mRNAs is then performed.

Alternatively, expression analysis using the PCTA-1 genomic DNA, a PCTA-1 cDNA, or fragments thereof can be done through high density nucleotide arrays as described by Lockhart et al. (1996) and Sosnowsky et al. (1997). Oligonucleotides of 15-50 nucleotides from the sequence of the PCTA-1 genomic DNA, a PCTA-1 cDNA sequence, particularly a sequence comprising at least one of biallelic markers according the present invention, preferably at least one of the biallelic markers A1 to A125 and the complements thereof or comprising the trait causing mutation, or a sequence complementary thereto, are synthesized directly on the chip (Lockhart et al., supra) or synthesized and then addressed to the chip (Sosnowski et al., supra). Preferably, the oligonucleotides are about 20 nucleotides in length.

PCTA-1 cDNA probes labeled with an appropriate compound, such as biotin, digoxigenin or fluorescent dye, are synthesized from the appropriate mRNA population and then randomly fragmented to an average size of 50 to 100 nucleotides. The said probes are then hybridized to the chip. After washing as described in Lockhart et al., supra and application of different electric fields (Sosnowsky et al., 1997)., the dyes or labeling compounds are detected and quantified. Duplicate hybridizations are performed. Comparative analysis of the intensity of the signal originating from cDNA probes on the same target oligonucleotide in different cDNA samples indicates a differential expression of PCTA-1 mRNA.

Screening for Molecules Interacting with a PCTA-1 Protein

The PCTA-1 proteins or fragments thereof described above may be used in drug screening procedures to identify molecules which are agonists, antagonists, or inhibitors of PCTA-1 activity. In a preferred embodiment, the PCTA-1 proteins or fragments thereof comprise at least one mutation provided either by biallelic markers of the present invention, preferably at least one mutation encoding by the biallelic markers A54, A56, A60, A75, A76, A85, or by a trait causing mutation according to the present invention. The PCTA-1 proteins or fragments thereof used in such analyses may be free in solution or linked to a solid support Alternatively, the PCTA-1 proteins or fragments thereof can be expressed on a cell surface. The cell may naturally express a PCTA-1 protein or a fragment thereof or, alternatively, the cell may express a PCTA-1 protein or a fragment thereof from an expression vector such as those described above.

In one method of drug screening, eucaryotic or procaryotic host cells which are stably transformed with recombinant polynucleotides in order to express a PCTA-1 protein or a fragment thereof are used in conventional competitive binding assays or standard direct binding assays.

To study the interaction of a PCTA-1 protein or a fragment thereof with drugs or small molecules, such as molecules generated through combinatorial chemistry approaches, the microdialysis coupled to HPLC method described by Wang et al. (1997) or the affinity capillary electrophoresis method described by Bush et al. (I 997) can be used.

In further methods, molecules which interact with a PCTA-1 protein or a fragment thereof may be identified using assays such as the following. The molecule to be tested for binding is labeled with a detectable label, such as a fluorescent, radioactive, or enzymatic tag and placed in contact with an immobilized PCTA-1 protein or a fragment thereof under conditions which permit specific binding to occur. After removal of non-specifically bound molecules, bound molecules are detected using appropriate means.

Another object of the present invention consists of methods and kits for the screening of candidate substances that interact with a PCTA-1 polypeptide.

A method for the screening of a candidate substance comprises the following steps: a) providing a polypeptide consisting of a PCTA-1 protein or a fragment thereof; b) obtaining a candidate substance; c) bringing into contact said polypeptide with said candidate substance; and d) detecting the complexes formed between said polypeptide and said candidate substance. Optionally, said PCTA-1 protein or fragment thereof is selected from the group consisting of polypeptides of SEQ ID Nos 5, 6, 7, 9 and fragments thereof.

The invention also pertains to kits useful for performing the hereinbefore described screening method. Preferably, such kits comprise a PCTA-1 polypeptide or a fragment thereof, and optionally means useful to detect the complex formed between a PCTA-1 polypeptide or a fragment thereof and the candidate substance. In a preferred embodiment the detection means consist in monoclonal or polyclonal antibodies directed against the corresponding PCTA-1 polypeptide or a fragment thereof.

Various candidate substances or molecules can be assayed for interaction with a PCTA-1 protein or a fragment thereof. These substances or molecules include, without being limited to, natural or synthetic organic compounds or molecules of biological origin such as polypeptides, antibodies, fatty acids and lipoproteins. When the candidate substance or molecule consists of a polypeptide, this polypeptide may be the resulting expression product of a phage clone belonging to a phage-based random peptide library, or alternatively the polypeptide may be the resulting expression product of a cDNA library cloned in a vector suitable for performing a two-hybrid screening assay.

For the purpose of the present invention, a ligand means a molecule, such as a protein, a peptide, an antibody, a fatty acid, a lipoprotein, or any synthetic chemical compound capable of binding to a PCTA-1 protein or a fragment thereof.

A. Candidate Ligands Obtained from Random Peptide Libraries

In a particular embodiment of the screening method, the putative ligand is the expression product of a DNA insert contained in a phage vector (Parm ley and Smith, 1988). Specifically, random peptide phages libraries are used. The random DNA inserts encode for peptides of 8 to 20 amino acids in length (Oldenburg K. R. et al., 1992; Valadon P., et al., 1996; Lucas A. H., 1994; Westerink M. A. J., 1995; Felici F. et al., 1991). According to this particular embodiment, the recombinant phages expressing a protein that binds to the immobilized PCTA-1 protein or a fragment thereof is retained and the complex formed between the PCTA-1 polypeptide and the recombinant phage may be subsequently immunoprecipitated by a polyclonal or a monoclonal antibody directed against the PCTA-1 polypeptide.

Once the ligand library in recombinant phages has been constructed, the phage population is brought into contact with the immobilized PCTA-1 protein or a fragment thereof. Then the preparation of complexes is washed in order to remove the non-specifically bound recombinant phages. The phages that bind specifically to the PCTA-1 protein or a fragment thereof are then eluted by a buffer (acid pH) or immunoprecipitated by the monoclonal antibody produced by the hybridoma anti-PCTA-1, and this phage population is subsequently amplified by an over-infection of bacteria (for example E. coli). The selection step may be repeated several times, preferably 2-4 times, in order to select the more specific recombinant phage clones. The last step consists of characterizing the peptide produced by the selected recombinant phage clones either by expression in infected bacteria and isolation, expressing the phage insert in another host-vector system, or sequencing the insert contained in the selected recombinant phages.

B. Candidate Ligands Obtained by Competition Experiments.

Alternatively, peptides, drugs or small molecules which bind to the PCTA-1 protein, or a fragment thereof may be identified in competition experiments. In such assays, the PCTA-1 protein or a fragment thereof is immobilized to a surface, such as a plastic plate. Increasing amounts of the peptides, drugs or small molecules are placed in contact with the immobilized PCTA-1 protein or a fragment thereof in the presence of a detectable labeled known PCTA-1 protein ligand. For example, the PCTA-1 ligand may be detectably labeled with a fluorescent, radioactive, or enzymatic tag. The ability of the test molecule to bind the PCTA-1 protein or a fragment thereof is determined by measuring the amount of detectably labeled known ligand bound in the presence of the test molecule. A decrease in the amount of known ligand bound to the PCTA-1 protein or a fragment thereof when the test molecule is present indicated that the test molecule is able to bind to the PCTA-1 protein or a fragment thereof.

C. Candidate Ligands Obtained by Affinity Chromatography.

Proteins or other molecules interacting with the PCTA-1 protein or a fragment thereof can also be found using affinity columns which contain the PCTA-1 protein or a fragment thereof. The PCTA-1 protein or a fragment thereof may be attached to the column using conventional techniques including chemical coupling to a suitable column matrix such as agarose, Affi Gel®, or other matrices familiar to those of skill in art. In some embodiments of this method, the affinity column contains chimeric proteins in which the PCTA-1 protein or a fragment thereof is fused to glutathion S transferase (GST). A mixture of cellular proteins or pool of expressed proteins as described above is applied to the affinity column. Proteins or other molecules interacting with the PCTA-1 protein or a fragment thereof attached to the column can then be isolated and analyzed on 2-D electrophoresis gel as described in Ramunsen et al. (1997). Alternatively, the proteins retained on the affinity column can be purified by electrophoresis based methods and sequenced. The same method can be used to isolate antibodies, to screen phage display products, or to screen phage display human antibodies.

D. Candidate Ligands Obtained by Optical Biosensor Methods

Proteins interacting with the PCTA-1 protein or a fragment thereof can also be screened by using an Optical Biosensor as described in Edwards and Leatherbarrow (1997) and also in Szabo et al. (1995). This technique permits the detection of interactions between molecules in real time, without the need of labeled molecules. This technique is based on the surface plasmon resonance (ISPR) phenomenon. Briefly, the candidate ligand molecule to be tested is attached to a surface (such as a carboxymethyi dextran matrix). A light beam is directed towards the side of the surface that does not contain the sample to be tested and is reflected by said surface. The SPR phenomenon causes a decrease in the intensity of the reflected light with a specific association of angle and wavelength. The binding of candidate ligand molecules cause a change in the refraction index on the surface, which change is detected as a change in the SPR signal. For screening of candidate ligand molecules or substances that are able to interact with the PCTA-1 protein or a fragment thereof, the PCTA-1 polypeptide is immobilized onto a surface. This surface consists of one side of a cell through which flows the candidate molecule to be assayed. The binding of the candidate molecule on the PCTA-1 protein or a fragment thereof is detected as a change of the SPR signal. The candidate molecules tested may be proteins, peptides, carbohydrates, lipids, or small molecules generated by combinatorial chemistry. This technique may also be performed by immobilizing eukaryotic or prokaryotic cells or lipid vesicles exhibiting an endogenous or a recombinantly expressed PCTA-1 protein at their surface.

The main advantage of the method is that it allows the determination of the association rate between the PCTA-1 protein and molecules interacting with the PCTA-1 protein. It is thus possible to select specifically ligand molecules interacting with the PCTA-1 protein, or a fragment thereof, through strong or conversely weak association constants.

E. Candidate Ligands Obtained through a Two-Hybrid Screening Assay.

The yeast two-hybrid system is designed to study protein-protein interactions in vivo (Fields and Song, 1989), and relies upon the fusion of a bait protein to the DNA binding domain of the yeast Gal4 protein. This technique is also described in the U.S. Patent N^(o) U.S. Pat. No. 5,667,973 and the U.S. Pat. N^(o) 5,283,173 (Fields et al.), the disclosures of which are incorporated herein by reference in their entireties.

The general procedure of library screening by the two-hybrid assay may be performed as described by Harperet al. (1993) or as described by Cho et al. (1998) or also Fromont-Racine et al. (1997).

The bait protein or polypeptide consists of a PCTA-1 polypeptide or a fragment thereof.

More precisely, the nucleotide sequence encoding the PCTA-1 polypeptide or a fragment thereof is fused to a polynucleotide encoding the DNA binding domain of the GAL4 protein, the fused nucleotide sequence being inserted in a suitable expression vector, for example pAS2 or pM3.

Then, a human cDNA library is constructed in a specially designed vector, such that the human cDNA insert is fused to a nucleotide sequence in the vector that encodes the transcriptional domain of the GAL4 protein. Preferably, the vector used is the pACT vector. The polypeptides encoded by the nucleotide inserts of the human cDNA library are termed “pray” polypeptides.

A third vector contains a detectable marker gene, such as beta galactosidase gene or CAT gene that is placed under the control of a regulation sequence that is responsive to the binding of a complete Gal4 protein containing both the transcriptional activation domain and the DNA binding domain. For example, the vector pG5EC may be used.

Two different yeast strains are also used. As an illustrative but non limiting example the two different yeast strains may be the followings:

Y190, the phenotype of which is (MATa, Leu2-3, 112 ura3-12, trp1-901, his3-D200, ade2-101, gal4Dga1180D URA3 GAL-LacZ, LYS GAL-HIS3, cyh');

Y187, the phenotype of which is (MaTa gal4 ga180 his3 trp1-901 ade2-101 ura3-52 leu2-3, -112 URA3 GAL-lacZmet′), which is the opposite mating type of Y190.

Briefly, 20 μg of pAS2/PCTA-1 and 20 μg of pACT-cDNA library are co-transformed into yeast strain Y190. The transformants are selected for growth on minimal media lacking histidine, leucine and tryptophan, but containing the histidine synthesis inhibitor 3-AT (50 mM). Positive colonies are screened for beta galactosidase by filter lift assay. The double positive colonies (His⁺, beta-gal⁺) are then grown on plates lacking histidine, leucine, but containing tryptophan and cycloheximide (10 mg/ml) to select for loss of pAS2/PCTA-1 plasmids bu retention of pACT-cDNA library plasmids. The resulting Y190 strains are mated with Y187 strains expressing PCTA-1 or non-related control proteins; such as cyclophilin B, lamin, or SNF1, as Gal4 fusions as described by Harper et al. (1 993) and by Bram et al. (1993), and screened for beta galactosidase by filter lift assay. Yeast clones that are beta gal- after mating with the control Gal4 fusions are considered false positives.

In another embodiment of the two-hybrid method according to the invention, interaction between the PCTA-1 or a fragment thereof with cellular proteins may be assessed using the Matchmaker Two Hybrid System 2 (Catalog No. K1604-1, Clontech). As described in the manual accompanying the Matchmaker Two Hybrid System 2 (Catalog No. K1604-1, Clontech), nucleic acids encoding the PCTA-1 protein or a fragment thereof, are inserted into an expression vector such that they are in frame with DNA encoding the DNA binding domain of the yeast transcriptional activator GAL4. A desired cDNA, preferably human cDNA, is inserted into a second expression vector such that they are in frame with DNA encoding the activation domain of GAL4. The two expression plasmids are transformed into yeast and the yeast are plated on selection medium which selects for expression of selectable markers on each of the expression vectors as well as GALA dependent expression of the HIS3 gene. Transformants capable of growing on medium lacking histidine are screened for GAL4 dependent lacZ expression. Those cells which are positive in both the histidine selection and the lacZ assay contain interaction between PCTA-1 and the protein or peptide encoded by the initially selected cDNA insert.

Screening through Spontaneous Metastatic Assay

Screening of new compounds can be carried out through a spontaneous metastatic assay as described in Nihei et al. (1995). Hence, it can be possible to assess the decrease of metastatic potential of transformed cells related to treatment of said compounds. Indeed, according to the present invention, the metastatic potential of cells constitutes the major criteria of the aggressiveness of prostate cancer tumors.

To evaluate the metastatic ability, about 5×10⁵ cells expressing a PCTA-1 gene comprising alleles for one or more biallelic markers associated with cancer, preferably with prostate cancer, or with the aggressiveness of prostate cancer tumors, are injected subcutaneously in the flank of male athymic nude mice. The mice are treated with the screened compounds. Tumor volume and tumor volume doubling time are used as the index of the tumor growth rate and are determined as described in Isaacs & Hukku, 1988). The tumor-bearing animals are scored for lung metastases at spontaneous death or when killed at day 35 post-inoculation.

Gene Therapy

Gene therapy involves the alteration of the phenotypic expression of a targeted cell, usually a cancer cell through the alteration of the cell's genotypic content. The desired effect of gene therapy is a reduction or interruption of tumor growth or, ideally, the destruction of the cell itself. An appropriate gene for gene therapy must be capable of altering the biological behavior of the cancer cell in order to slow growth, reduce local invasive potential, or induce apoptosis. The PCTA-1 gene, or certain portions thereof, is a good candidate for gene therapy.

The present invention also comprises the use of the genomic PCTA-1 DNA described above or a fragment thereof, in gene therapy strategies, such as antisense and triple helix strategies, and in the introduction of a therapeutic gene. Preferred nucleotide sequences useful in gene therapy include the sequences of SEQ ID Nos 1, 2, 3, 4, 8, complementary sequences thereto, and fragments thereof. More particularly, preferred nucleotide sequences comprise any of the polynucleotides described in the “PCTA-1 cDNA Sequences” section, the “Coding Regions” section, and the “Oligonucleotide Probes And Primers” section. Preferred PCTA-1 DNA fragments used in such approaches are those comprising a nucleotide sequence comprising a PCTA-1 regulatory region or a fragment thereof. More particularly, the regulatory regions comprise at least one of the biallelic markers according to the invention, more particularly those comprising a biallelic marker selected from the group consisting of A1 to A125, preferably A2, A30, A41, A55, A57, or a trait causing mutation, or complementary sequences thereof or variants or fragments thereof.

In a first embodiment, the invention therefore concerns a method for the treatment of prostate cancer. The method comprises: (a) selecting an individual whose DNA comprises an allele of biallelic marker or of a group of biallelic markers, preferably markers of the PCTA-1 gene, associated with a susceptibility to prostate cancer; and (b) administering to the individual an effective amount of a molecule capable of modifying the expression of the PCTA-1 gene:

In one embodiment, the molecule is an antisense nucleotide sequence, capable of competitively binding to the mRNA sequence resulting from the transcription of the PCTA-1 genomic DNA so as to prevent the translation of said mRNA. In preferred embodiments of this method, the antisense nucleotide sequence is characterized in that it hybridizes with exons of the PCTA-1 gene, preferably with a region of such exons comprising a least an allele of one of the biallelic markers of the present invention. Optionally, the antisense nucleotide sequence hybridizes with exons 0, 1, 6bis, 9 or 9ter of the PCTA-1 gene.

In an other embodiment, the molecule is a nucleotide sequence comprising a homopurine or homopyridine, preferably a 10-mer to 20-mer homopurine or homopyridine, which is complementary to a homopurine or homopyridine sequence of the PCTA-1 genomic DNA so as to prevent transcription of said genomic DNA into mRNA.

In a further embodiment, the molecule is a nucleotide sequence comprising a DNA sequence encoding a protein capable, when expressed, of exerting a therapeutic effect on prostate cancer cells, operably linked to the promoter of PCTA-1 gene, so as to kill or disable said prostate cancer cells.

The invention also concerns a method for the treatment of prostate cancer comprising:

administering to an individual an effective amount of a nucleotide sequence comprising a DNA sequence encoding a protein capable, when expressed, of exerting a therapeutic effect on prostate cancer cells, operably linked to the promoter of PCTA-1 gene.

The gene encoding a protein capable of exerting a therapeutic effect on prostate cancer cells is called the therapeutic gene in the present application. In some embodiments, the therapeutic gene is a toxin gene encoding a cytotoxic or cytostatic gene product. In another embodiment, the therapeutic gene is a gene encoding an immunogenic antigen which is highly visible to the immune system. In further embodiment, the therapeutic gene is a gene encoding a lymphokine which activates an anti-tumor immune response. In additional embodiments, the therapeutic gene encodes an antisense sequence having as a target the coding region of an essential gene for the proliferation or viability of the cell.

Antisense Approach

In antisense approaches, nucleic acid sequences complementary to a targeted mRNA are hybridized to the mRNA intracellularly, thereby blocking the expression of the protein encoded by the mRNA. The antisense sequences can prevent gene expression through a variety of mechanisms. For example, the antisense sequences may inhibit the ability of ribosomes to translate the mRNA. Alternatively, the antisense sequences may block transport of the mRNA from the nucleus to the cytoplasm, thereby limiting the amount of mRNA available for translation. Another mechanism through which antisense sequences may inhibit gene expression is by interfering with mRNA splicing. In yet another strategy, the antisense nucleic acid may be incorporated in a ribozyme capable of specifically cleaving the target mRNA.

The antisense nucleic acid molecules to be used in gene therapy may be either DNA or RNA sequences. They comprise a nucleotide sequence complementary to the targeted sequence of the PCTA-1 genomic DNA or a PCTA-1 cDNA. The targeted DNA or RNA sequence preferably comprises at least one of the biallelic markers according to the present invention, particularly a biallelic marker selected from the group consisting of A1 to A125 and the complements thereof, or comprises a trait causing mutation. In a preferred embodiment, the antisense oligonucleotide are able to hybridize with at least one of the splicing sites of the targeted PCTA-1 gene, with the 3′UTR or the 5′UTR, with exon 0, 1, 6bis, 9 or 9ter, or with an exonic region comprising at least one of the biallelic markers of the present invention or comprising a trait causing mutation.

Preferred methods using antisense polynucleotide according to the present invention are the procedures described by Sczakiel et al.(1995).

Preferably, the antisense tools are chosen among the polynucleotides (15-200 bp long) that are complementary to the 5′ end of the PCTA-1 mRNA. In another embodiment, a combination of different antisense polynucleotides complementary to different parts of the desired targeted gene are used.

Preferred antisense polynucleotides according to the present invention are complementary to a sequence of the mRNAs of PCTA-1 that contains either the translation initiation codon ATG or a splicing donor or acceptor site.

The antisense nucleic acids should have a length and melting temperature sufficient to permit formation of an intracellular duplex having sufficient stability to inhibit the expression of the PCTA-1 mRNA in the duplex. Strategies for designing antisense nucleic acids suitable for use in gene therapy are disclosed in Green et al., (1986) and Izant and Weintraub, (1984).

In some strategies, antisense molecules are obtained by reversing the orientation of the PCTA-1 coding region with respect to a promoter so as to transcribe the opposite strand from that which is normally transcribed in the cell. The antisense molecules may be transcribed using in vitro transcription systems such as those which employ T7 or SP6 polymerase to generate the transcript. Another approach involves transcription of PCTA-1 antisense nucleic acids in vivo by operably linking DNA containing the antisense sequence to a promoter in a suitable expression vector.

Alternatively,suitable antisense strategies are those described by Rossi et al.(1991), in the International Applications Nos. WO 94/23026, WO 95/04141, WO 92/18522, WO 96/31523 and in the European Patent Application No. EP 0 572 287 A2, the disclosures of which are incorporated herein by reference in their entireties.

An alternative to the antisense technology that is used according to the present invention consists of using ribozymes that will bind to a target sequence via their complementary polynucleotide tail and that will cleave the corresponding RNA by hydrolyzing its target site (namely “hammerhead ribozymes”). Briefly, the simplified cycle of a hammerhead ribozyme consists of (1) sequence specific binding to the target RNA via complementary antisense sequences; (2) site-specific hydrolysis of the cleavable motif of the target strand; and (3) release of cleavage products, which gives rise to another catalytic cycle. Indeed, the use of long-chain antisense polynucleotide (at least 30 bases long) or ribozymes with long antisense arms are advantageous. A preferred delivery system for antisense ribozyme is achieved by covalently linking these antisense ribozymes to lipophilic groups or to use liposomes as a convenient vector. Preferred antisense ribozymes according to the present invention are prepared as described by Sczakiel et al.(1995).

Triple Helix Approach

The PCTA-1 genomic DNA, preferably comprising at least one of the biallelic markers according to the invention, more preferably at least one biallelic marker selected from the group consisting of A1 to A125, or comprising a trait causing mutation, or complementary sequences, variants or fragments thereof, may also be used in gene therapy approaches based on intracellular triple helix formation.

Triple helix oligonucleotides are used to inhibit transcription from a genome. They are particularly useful for studying alterations in cell activity when it is associated with a particular gene. Fragments of the PCTA-1 genomic DNA can be used to inhibit gene expression in individuals suffering from prostate cancer or from another detectable phenotype, or in individuals at risk of developing prostate cancer or another detectable phenotype at a later date as a result of their PCTA-1 genotype.

Similarly, a portion of the PCTA-1 genomic DNA can be used to study the effect of inhibiting PCTA-1 transcription within a cell. Traditionally, homopurine sequences were considered the most useful for triple helix strategies. However, homopyrimidine sequences can also inhibit gene expression. Such homopyrimidine oligonucleotides bind to the major groove at homopurine:homopyrimidine sequences. Thus, both types of sequences from the PCTA-1 genomic DNA, preferably comprising at least one of the biallelic markers according to the invention, more preferably at least one of the biallelic markers A1 to A125, or comprising the trait causing mutation, or complementary sequences thereof, variants thereof, are contemplated within the scope of this invention.

To carry out gene therapy strategies using the triple helix approach, the sequences of the PCTA-1 genomic DNA, preferably comprising at least one of the biallelic markers according to the invention, or comprising the trait causing mutation, or complementary sequences thereof, or variants thereof, are first scanned to identify 10-mer to 20-mer homopyrimidine or homopurine stretches which could be used in triple-helix based strategies for inhibiting PCTA-1 expression. Following identification of candidate homopyrimidine or homopurine stretches, their efficiency in inhibiting PCTA-1 expression is assessed by introducing varying amounts of oligonucleotides containing the candidate sequences into tissue culture cells which express the PCTA-1 gene.

The oligonucleotides can be introduced into the cells using a variety of methods known to those skilled in the art, including but not limited to calcium phosphate precipitation, DEAE-Dextran, electroporation, liposome-mediated transfection or native uptake.

Treated cells are monitored for altered cell function or reduced PCTA-1 expression using techniques such as Northern blotting, RNase protection assays, or PCR based strategies to monitor the transcription levels of the PCTA-1 gene in cells which have been treated with the oligonucleotide.

The oligonucleotides which are effective in inhibiting gene expression in tissue culture cells may then be introduced in vivo using the techniques described above in the antisense approach at a dosage calculated based on the in vitro results, as described in antisense approach.

In some embodiments, the natural (beta) anomers of the oligonucleotide units can be replaced with alpha anomers to render the oligonucleotide more resistant to nucleases. Further, an intercalating agent such as ethidium bromide, or the like, can be attached to the 3′ end of the alpha oligonucleotide to stabilize the triple helix. For information on the generation of oligonucleotides suitable for triple helix formation see Griffin et al. (1989).

Introduction of a Therapeutic Gene

One important aspect of the present invention concerns a promoter specifically expressed in prostate cancer cells. More particularly, the present invention relates to the regulatory sequences, and particularly the promoter of the PCTA-1 gene. The expression of PCTA-1 appears to be specific to prostate cancer cells.

The term “specific”, when used herein with reference to a promoter, is intended to designate a promoter which is specifically expressed in prostate cancer cells, at a level which is sufficient to have a significant impact on the metabolism of such cells. In other words, the promoter is specific in activity, effect or function. However, the term does not necessarily designate a promoter which is expressed solely in prostate cancer cells. Indeed, it is possible that the PCTA-1 gene is expressed, under the control of its promoter, in other cells at levels which are sufficiently low to be undetectable by current detection techniques such as those involving antibodies, hybridization with a probe or even PCR. The promoter of the PCTA-1 gene can be advantageously used to introduce a therapeutic gene which will be expressed specifically in prostate cancer cells.

The invention therefore also concerns an expression vector comprising a DNA sequence encoding a functional protein, particularly a functional protein capable of exerting a therapeutic effect on prostate cancer cells, operably linked to the promoter of the PCTA-1 gene which is specifically expressed in prostate cancer cells.

Furthermore, the PCTA-1 promoter preferably comprises biallelic markers according to the invention, more particularly those described previously. Some alleles of the biallelic markers of the invention show an association with prostate cancer and may be involved in a modified or forthcoming expression of the PCTA-1 gene in prostate cancer cells. It may therefore advantageous to use the PCTA-1 promoter comprising such an allele to introduce a therapeutic gene for enhancing its expression in prostate cancer cells.

The term “therapeutic gene” is intended to designate DNA encoding an amino acid sequence corresponding to a functional peptide or protein capable of exerting a therapeutic effect on prostate cancer cells preferably by killing or disabling such cells, or having a regulatory effect on the expression of an important function in prostate cells.

In one embodiment, a single enhancer element or multiple enhancer elements which amplify the expression of the therapeutic gene without compromising tissue specificity can also be combined with the promoter of the PCTA-1 gene. In a preferred embodiment, the enhancer element may be a portion of the cytomegalovirus LTR, SV40 enhancer sequences, or MMTV LTR. Preferably, the enhancer element is positioned upstream of the PCTA-1 promoter.

The term “enhancer element” is intended to designate a nucleotide sequence that increases the rate of transcription of therapeutic genes or genes of interest but does not have promoter activity. An enhancer can be moved upstream, downstream, and to the other side of the PCTA-1 promoter without significant loss of activity.

In a preferred embodiment, a vector is constructed by inserting the therapeutic gene downstream of the PCTA-1 promoter. The therapeutic gene is inserted so as to be operably linked to the promoter.

Examples of therapeutic genes include suicide genes. These are gene sequences, the expression of which produces a protein or agent that inhibits prostate tumor cell growth or induces prostate tumor cell death. Genes of interest include genes encoding enzymes, oncogenes, tumor suppressor genes, genes encoding toxins, genes encoding cytokines, or a gene encoding oncostatin. The purpose of the therapeutic genes is to inhibit the growth of or kill prostate cancer cells or to produce cytokines or other cytotoxic agents which directly or indirectly inhibit the growth of or kill prostate cancer cell.

Suitable enzymes include thymidine kinase, xanthine-guanine phosphoribosyltransferase, cytosine deaminase, and hypoxanthine phosphoribosyl transferase. Suitable oncogenes and tumor suppressor genes include neu, EGF, ras, p53, retinoblastoma tumor suppressor gene (Rb), Wilm's tumor gene product, phosphotyrosine phosphatase, and nm23. Suitable toxins include Pseudomonas exotoxin A and S, diphteria toxin, E. coli LT toxins, Shiga toxin, Shiga-like toxins, ricin, abrin, supporin, and gelonin. Suitable cytokines include interferons, GM-CSF interleukins, tumor necrosis factor.

Other gene therapy strategies include antisense sequences as mentioned above of at least about 30 bp, preferably 50 pb, having as target the coding sequence of an essential gene for the proliferation or viability of the cell. Numerous proteins associated with transcription, translation, metabolic pathways, cytostructural genes can be used as target, preferably those which are essential, present at relatively low levels, and particularly associated with cancer cells.

The three presently available methodologies for DNA delivery are well-known by the skilled artisan: transfection with a viral vector, fusion with a lipid; and cationic supported DNA introduction. A suitable DNA delivery method should meet the following criteria: 1) capable of directing the therapeutic polynucleotides into specific target cell types, 2) highly efficient in mediating uptake of the therapeutic polynucleotide into the target cells, and 3) suited for use in vivo for therapeutic application.

The preferred method relies on replication-defective viral vectors harboring the therapeutic polynucleotide sequence as part of retroviral genome. Preferred vectors for use in the present invention are viral including adenoviruses, retroviral vectors, and adeno-associated viral vectors. Retroviral vectors and adenoviruses offer an efficient, useful, and presently the best-characterized means of introducing and expressing foreign genes efficiently in mammalian cells. These vectors have very broad host and cell type range and express genes stably and efficiently.

Other virus vectors that may be used for gene transfer into cells include retroviruses such as Moloney murine lekemia virus, papovaviruses such as JC, SV40, polyoma, and adenoviruses, Epstein-Barr virus, papilloma viruses such as bovine papilloma virus type I, vaccinia, and poliovirus.

Another gene transfer method is physical transfer of plasmid DNA comprising the therapeutic polynucleotide in liposomes directly into prostate, preferably into tumors cells in situ. Immunoliposomes may improve cell type specificity as compared to liposomes by virtue of the inclusion of specific antibodies which presumably bind to surface antigens specific of prostate cells. In one embodiment, antibodies are directed against PCTA-1 protein which is specific to prostate cancer cells.

Direct physical application of naked DNA comprising the therapeutic polynucleotide to the target cells is believed to be preferred in many cases.

Vaccine Composition

The invention concerns a vaccine composition comprising a vaccination agent including one of the following polypeptide:

a) a polypeptide comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15,20, 25,30,40, 50, or 100 amino acids of SEQ ID No 5, wherein said contigous span comprises:

i) a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 203 in SEQ ID No 5; and/or

ii) at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 183 in SEQ ID No 5;

b) a polypeptide comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25,30,40, 50, or 100 amino acids of SEQ ID No 6, wherein said contigous span comprises:

i) a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 245 in SEQ ID No 6; and/or

ii) at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 225 in SEQ ID No 6; and/or

iii) at least 1, 2, 3, 5 or 10 of the amino acid encoded by the exon 6bis, more CF particularly at least 1, 2, 3, 5 or 10 of the amino acid positions 183-224 of the SEQ ID No 6;

c) a polypeptide comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20,25,30, 40, 50, or 100 amino acids of SEQ ID No 7, wherein said contigous span comprises:

i) a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 203 in SEQ ID No 7; and/or

ii) at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 183 in SEQ ID No 7; and/or

iii) at least 1, 2, 3, 5 or 10 of the amino acid encoded by the exons 9bis and 9ter, more particularly at least 1, 2, 3, 5 or 10 of the amino acid positions 313-368 of the SEQ ID No 7; and

d) a polypeptide comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25,30, 40, 50, or 100 amino acids of SEQ ID No 9.

“Vaccine agent or vaccination agent” is intended to designate a substance which has the ability, when administered to a patient in suitable amounts, to generate an immunogenic reaction which can confer either immunity to the patent against prostate cancer or kill or disable prostate cancer cells bearing on their surface the PCTA-1 protein or a fragment thereof.

The vaccine compositions of the present invention are intended to be administered to patients in an amount sufficient to inhibit the growth of cancer cells expressing the PCTA-1 protein. More particularly the vaccine composition is intended to decrease the growth rate, rate of division, or viability of the prostate cancer cells.

The administration of a vaccine composition of the invention may be for either a “prophylactic” or “therapeutic” purposes. When provided prophylactically, the vaccine agent are provided in advance of symptoms indicative of prostate cancer. The prophylactic administration of vaccine agent serves to prevent, attenuate, or inhibit of the growth of prostate cancer cells. The therapeutic administration of the vaccine agent serves to attenuate the pathological symptoms of prostate cancer, to decrease the size or growth of cancer tumors and or metastasis or to remove them.

The term “inhibition of growth” refers in the present application to the decrease of the rate of growth, rate of division, or viability of the cells in question.

Indeed, as the PCTA-1 gene is specifically expressed in prostate cancer cells, these vaccine agents can initiate the production of PCTA-1 specific cytotoxic T lymphocytes which lyse cells bearing, preferably on their surface, PCTA-1, a fragment of PCTA-1, or one or more PCTA-1 epitope peptides thereof and which lead to an inhibition of the growth of cancer also bearing the PCTA-1 protein.

Vaccine preparations which contain protein or peptide sequences as active substances are generally well known in the art, as exemplified by U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, the disclosures of which are incorporated herein by reference in their entireties.

A vaccine according to the present invention may further contain auxiliary vaccine constituents, such as carriers, buffers, stabilizers, solubilizers, adjuvants and preservatives.

In order to enhance the immunogenic character of the polypeptides taken from the mutated PCTA-1 protein, the polypeptides may be prepared as homopolymers (a multitude of identical polypeptides coupled to one another) or heteropolymers (a multitude of at least two different polypeptides coupled to one another).

The vaccine agents of the present invention can be used in native form or can be modified to form a chemical derivative. As used herein, a molecule is said to be a “chemical derivative” of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties may improve the molecule's solubility, absorption, biological half life, etc. The moieties may alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, et. Moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences (1980).

The vaccine agents of the present invention may be administered in a convenient manner such as by oral, topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, or intradermal routes. The vaccine agents of the present invention are administered in an amount which is effective for treatment and/or prophylaxis of the specific indication. In general, they are administered in an amount of at least about 10 μg/kg body weight per day and in most cases they are administered in an amount not in excess of about 8 mg/kg body weight per day. In most cases, the dosage is from about 10 μg/kg to about 1 mg/kg body weight daily, taking into account the routes of administration, symptoms, etc.

When administering the vaccine agent of the present invention to a patient, the dosage of the administered vaccine agent varies depending upon such factors as the patient's age, weight, sex, general medical condition, previous medical history. In general, it is desirable to provide the recipient with a dosage of vaccine agent which is in the range of from about 1 pg/kg to 10 mg/kg body weight, although a lower or higher dosage may be administered. The therapeutically effective dose can be lowered by using combinations of the vaccine agents of the present invention or other agents.

It is normally necessary to have multiple administrations of the vaccine agents, usually not exceeding six vaccinations, more usually not exceeding four vaccinations, preferably one or more vaccinations, more preferably about three vaccinations. The vaccinations will be normally be at from two to twelve week intervals, more usually from three to five week intervals. Periodic boosters at intervals of 1-5 years will be desirable to maintain levels of protective immunity.

The vaccine agents of the present invention are intended to be provided to recipient subjects in an amount sufficient to inhibit the growth (as defined above) of cancer cells bearing PCTA-1 protein.

The effect of the vaccine agents of the present invention can be assessed through the measurement of released IFN-γ from memory T-lymphocytes. The stronger of the immune response, the more IFN-γ will be released. Accordingly, a vaccine according to the invention comprises a polypeptide capable of releasing from the memory T-lymphocytes at least 1500 pg/ml, preferably 200 pg/ml, and more preferably 300 pg/ml of IFN-γ. Practically, the levels of IFN-γ from the primed lymphocytes are measured with in vitro proliferation assays of peripheral blood lymphocytes co-cultured with a vaccine agents to be tested. These techniques are well known and may be found in a wide variety of patents, such as U.S. Pat. No. 3,791,932; 4,174,384; and 3,949,064, the disclosures of which are incorporated herein by reference in their entireties, as illustrative of these types of assays.

The administration of the vaccine agent of the invention may be for either a “prophylactic” or “therapeutic” purposes. When provided prophylactically, the vaccine agent are administered in advance of any symptoms indicative of prostate cancer. The prophylactic administration of the vaccine agent serves to prevent, attenuate, or inhibit of the growth of prostate cancer cells. The therapeutic administration of the vaccine agent serves to attenuate the pathological symptoms of prostate cancer and to decrease the size of prostate cancer tumors or to remove them.

Typically, such vaccine agents are prepared as injectable either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may be emulsified. The active immunogenic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the vaccine agent. Suitable excipients are, for example, water, saline, dextrose, ethanol, or the like, and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccines.

PCTA-1 protein and peptides, preferably mutated, may be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts which are formed between the free amino groups of the peptide, and inorganic acids, such as hydrochloric or phosphoric acids, or organic acids, such as acetic oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxydes, or from organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

Some of the polypeptides of the vaccine agents of the invention are sufficiently immunogenic in a vaccine, but the immune response can be enhanced if the vaccine further comprises an adjuvant substance.

Various methods of achieving adjuvant effects for vaccines include the use of agents such as aluminim hydroxide or phosphate, commonly used as 0.05 to 0.1 percent solution in phosphate buffered saline, admixture with synthetic polymers of sugars (Carbopol) used as 0.25 percent solution, aggregation of the protein in the vaccine by heat treatment with temperature ranging between 70° C. and 101 C for 30 second to 2 minute periods, respectively. Aggregation by reacting with pepsin treated antibodies (Fab) to albumin, mixture with bacterial cells such as C. parvum or endotoxins or lipopolysaccharide components of gram-negative bacteria, emulsion in physiologically acceptable oil vehicles such as mannide monoleatc (Aracel A) or emulsion with 20 percent solution of a perfluorocarbon (Fluosol-DA) used as a block substitute may also be employed. According to the invention, dimethyldioctadecylammonium bromide is an interesting candidate for an adjuvant, but also Freund's complete and incomplete adjuvants as well as QuilA and RIBI are interesting possibilities. Other possibilities involve the use of immune modulating substances such as lymphokines (e.g. IFN-γ, IL-2 and IL-12) or synthetic IFN-γ inducers such as poly I:C in combination with the above-mentioned adjuvants.

The vaccine agent of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby immunogenic peptides, or their functional derivatives, are combined in admixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation, inclusive of other human proteins, such as human serum albumin, are described Remington's Pharmaceutical Sciences (1980). In order to form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of one or more of the vaccine agents of the present invention, together with a suitable amount of a carrier vehicle.

Additional pharmaceutical methods may be employed to control the duration of action. Control release preparations may be achieved through the use of polymers to complex or absorb one or more of the vaccine agents of the present invention. The controlled delivery may be exercised by selecting appropriate macromolecule (for example polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, protamine, or sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release. Another possible method to control the duration of action by controlled release preparations is to incorporate vaccine agents of the present invention into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers. Alternatively, instead of incorporating these vaccine agents into polymeric particles, it is possible to entrap these materials in microcapsules prepared, for example, bu coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatine-microcapsules and poly(methylmethacylate) microcapsules, respectively, orin colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (1980).

The invention further provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the vaccine compositions of the invention.

Computer-Related Embodiments

As used herein the term “nucleic acid codes of the invention” encompass the nucleotide sequences comprising, consisting essentially of, or consisting of any one of the following: a) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 1: 1-70715, 70795-82207, 82297-83612, 83824-85297, 85418-86388, 86446-87495, 87523-88294, 88384-89483, 89650-92748, 97156-98309, 98476-99329, 99491-100026, 100212-100281, 100396-100538, 100682-100833, 100995-101920, 102087-102970, 103264-103724, and 103753-106746; b) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200,500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide G at positions 70728, 87860, 88297, 94432, and 95340 of SEQ ID No 1; a nucleotide A at positions 82218, 83644, 83808, 87787, 87806, 94218, and 97144 of SEQ ID No 1; a nucleotide C at positions 87902, 88215, 88283, 92760,93726, and 94422 of SEQ ID No 1; and a nucleotide T at positions 93903, and 94170 of SEQ ID No 1; c)a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide G at positions 86435, 93592, 93680, 93681, 93682, 93728, 93761, and 95445 of SEQ ID No 1; a nucleotide A at positions 86434, 88355, 93240, 93471, and 93747of SEQ ID No 1; a nucleotide C at positions 93683, 95126, and 95444 of SEQ ID No 1; and a nucleotide T at positions 94154, and 94430 of SEQ ID No 1; d) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises nucleotide positions selected from the group consisting of the nucleotide positions of SEQ ID No 1: 92975-92977, 93711-93715,94151-94153, 94240-94243, 94770-94773, 94804-94808, 95121-95122, 95129-95135,95148-95153, 95154-95159, 95173-95178, 95367-95374, 95410-95413, 95418-95420, 95430-95436, 95533-95535, and 95677-95677; e) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the nucleotide positions 1-162 of SEQ ID No 2; f) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 253, 363, 527, 2471, and 5397 of SEQ ID No 2; a nucleotide C at positions 1013, 1979, and 2675 of SEQ ID No 2; a nucleotide G at positions 176, 749, 2685, 3593 of SEQ ID No 2; and a nucleotide T at positions 2156, and 2423 of SEQ ID No 2; g) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 708, 807, 1493, 1724, and 2000; a nucleotide C at positions 1936, 3379, and 3697; a nucleotide G at positions 709, 1845, 1933, 1934, 1935, 1981, 2014, and3698; and a nucleotide T at positions 2407, and 2683 of SEQ ID No 2; h) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises nucleotide positions selected from the group consisting of the nucleotide positions of SEQ ID No 2: 1229-1231, 1964-1968, 2404-2406, 2493-2496, 3023-3026, 3057-3061, 3374-3375, 3382-3388, 3401-3406, 3407-3412, 3426-3431, 3620-3627, 3663-3666, 3671-3673, 3683-3689, 3786-3788 and 3930-3932; i) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3,5, or 10 of the following nucleotide positions of SEQ ID No 3: 1-162 and 747-872; j) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 253, 363, 527, 2597, and 5523 of SEQ ID No 3; a nucleotide C at positions 1139, 2105, and 2801 of SEQ ID No 3; a nucleotide G at positions 176, 875, 2811, 3719 of SEQ ID No 3; and a nucleotide T at positions 2282, and 2549 of SEQ ID No 3; k) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 708, 807, 1619, 1850, and 2126; a nucleotide C at positions 2062, 3505, and 3823; a nucleotide G at positions 709, 1971, 2059, 2060, 2061, 2107, 2140, and 3824; and a nucleotide T at positions 2533, and 2809 of SEQ ID No 3; 1) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises nucleotide positions selected from the group consisting of the nucleotide positions of SEQ ID No 3: 1355-1357, 1892-1894, 2090-2094, 2530-2532, 2619-2622, 3149-3152, 3183-3187, 3500-3501, 3508-3514, 3527-3532, 3533-3538, 3552-3557, 3746-3749, 3789-3792, 3797-3799, 3809-3815, 3912-3914 and 4056-4058; m) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 4 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the nucleotide positions 1-162 of SEQ ID No 4; n) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 4 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 253, 363, 527 and 2460 of SEQ ID No 4; a nucleotide C at position 1013 of SEQ ID No 4 and a nucleotide G at positions 176, and 749 of SEQ ID No 4; o) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 4 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 708 and 807 and a nucleotide G at position 709 of SEQ ID No 4; p) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 4 or the complements thereof, wherein said contiguous span comprises the pairs of nucleotide positions 1136-1137 of SEQ ID No 4; q) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 8 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 8: 1-500, 501-1000, 1001-1500, and 1501-1738; and, r) a nucleotide sequence complementary to any one of the preceding nucleotide sequences.

The “nucleic acid codes of the invention” further encompass nucleotide sequences homologous to: a) a contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 1: 1-70715, 70795-82207, 82297-83612, 83824-85297, 85418-86388, 86446-87495, 87523-88294, 88384-89483, 89650-92748, 97156-98309, 98476-99329, 99491-100026, 100212-100281, 100396-100538, 100682-100833, 100995-101920, 102087-102970, 103264-103724, and 103753-106746; b) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the nucleotide positions 1-162 of SEQ ID No 2; c) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 3: 1-162 and 747-872; d) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 4 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the nucleotide positions 1-162 of SEQ ID No 4; e) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 8 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 8: 1-500, 501-1000, 1001-1500, and 1501-1738; and f) sequences complementary to all of the preceding sequences. Homologous sequences refer to a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, or 75% homology to these contiguous spans. Homology may be determined using any method described herein, including BLAST2N with the default parameters or with any modified parameters. Homologous sequences also may include RNA sequences in which uridines replace the thymines in the nucleic acid codes of the invention. It will be appreciated that the nucleic acid codes of the invention can be represented in the traditional single character format (See the inside back cover of Stryer, Lubert. Biochemistry, 3^(rd) edition. W. H Freeman & Co., New York.) or in any other format or code which records the identity of the nucleotides in a sequence.

As used herein the term “polypeptide codes of the invention” encompass the polypeptide sequences comprising:

a) a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 5, wherein said contiguous span includes:

i) a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 203 in SEQ ID No 5; and/or

ii) at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 183 in SEQ ID No 5;

b) a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 6, wherein said contiguous span includes:

i) a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 245 in SEQ ID No 6; and/or

ii) at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 225 in SEQ ID No 6; and/or

iii) at least 1, 2, 3, 5 or 10 of the amino acid encoded by the exon 6bis, more particularly at least 1, 2, 3, 5 or 10 of the amino acid positions 183-224 of the SEQ ID No 6;

c) a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 7, wherein said contiguous span includes:

i) a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 203 in SEQ ID No 7; and/or

ii) at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 183 in SEQ ID No 7; and/or

iii) at least 1, 2, 3, 5 or 10 of the amino acid encoded by the exons 9bis and 9ter, more particularly at least 1, 2, 3, 5 or 10 of the amino acid positions 313-368 of the SEQ ID No 7; and,

d) a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 9.

It will be appreciated that the polypeptide codes of the invention can be represented in the traditional single character format or three letter format (See the inside back cover of Stryer, Lubert. Biochemistry, 3^(rd) edition. W. H Freeman & Co., New York.) or in any other format or code which records the identity of the polypeptides in a sequence.

It will be appreciated by those skilled in the art that the nucleic acid codes of the invention and polypeptide codes of the invention can be stored, recorded, and manipulated on any medium which can be read and accessed by a computer. As used herein, the words “recorded” and “stored” refer to a process for storing information on a computer medium. A skilled artisan can readily adopt any of the presently known methods for recording information on a computer readable medium to generate manufactures comprising one or more of the nucleic acid codes of the invention, or one or more of the polypeptide codes of the invention. Another aspect of the present invention is a computer readable medium having recorded thereon at least 2, 5, 10, 15, 20, 25, 30, or 50 nucleic acid codes of the invention. Another aspect of the present invention is a computer readable medium having recorded thereon at least 2, 5, 10, 15, 20, 25, 30, or 50 polypeptide codes of the invention.

Computer readable media include magnetically readable media, optically readable media, electronically readable media and magnetic/optical media. For example, the computer readable media may be a hard disk, a floppy disk, a magnetic tape, CAROM, Digital Versatile Disk (DVD), Random Access Memory (RAM), or Read Only Memory (ROM) as well as other types of other media known to those skilled in the art.

Embodiments of the present invention include systems, particularly computer systems which store and manipulate the sequence information described herein. One example of a computer system 100 is illustrated in block diagram form in FIG. 3. As used herein, “a computer system” refers to the hardware components, software components, and data storage components used to analyze the nucleotide sequences of the nucleic acid codes of the invention or the amino acid sequences of the polypeptide codes of the invention. In one embodiment, the computer system 100 is a Sun Enterprise 1000 server (Sun Microsystems, Palo Alto, Calif.). The computer system 100 preferably includes a processor for processing, accessing and manipulating the sequence data. The processor 105 can be any well-known type of central processing unit, such as the Pentium III from Intel Corporation, or similar processor from Sun, Motorola, Compaq or International Business Machines.

Preferably, the computer system 100 is a general purpose system that comprises the processor 105 and one or more internal data storage components 110 for storing data, and one or more data retrieving devices for retrieving the data stored on the data storage components. A skilled artisan can readily appreciate that any one of the currently available computer systems are suitable.

In one particular embodiment, the computer system 100 includes a processor 105 connected to a bus which is connected to a main memory 115 (preferably implemented as RAM) and one or more internal data storage devices 110, such as a hard drive and/or other computer readable media having data recorded thereon. In some embodiments, the computer system 100 further includes one or more data retrieving device 118 for reading the data stored on the internal data storage devices 110.

The data retrieving device 118 may represent, for example, a floppy disk drive, a compact disk drive, a magnetic tape drive, etc. In some embodiments, the internal data storage device 110 is a removable computer readable medium such as a floppy disk, a compact disk, a magnetic tape, etc. containing control logic and/or data recorded thereon. The computer system 100 may advantageously include or be programmed by appropriate software for reading the control logic and/or the data from the data storage component once inserted in the data retrieving device.

The computer system 100 includes a display 120 which is used to display output to a computer user. It should also be noted that the computer system 100 can be linked to other computer systems 125 a-c in a network or wide area network to provide centralized access to the computer system 100.

Software for accessing and processing the nucleotide sequences of the nucleic acid codes of the invention or the amino acid sequences of the polypeptide codes of the invention (such as search tools, compare tools, and modeling tools etc.) may reside in main memory 115 during execution.

In some embodiments, the computer system 100 may further comprise a sequence comparer for comparing the above-described nucleic acid codes of the invention or the polypeptide codes of the invention stored on a computer readable medium to reference nucleotide or polypeptide sequences stored on a computer readable medium. A “sequence comparer” refers to one or more programs which are implemented on the computer system 100 to compare a nucleotide or polypeptide sequence with other nucleotide or polypeptide sequences and/or compounds including but not limited to peptides, peptidomimetics, and chemicals stored within the data storage means. For example, the sequence comparer may compare the nucleotide sequences of nucleic acid codes of the invention or the amino acid sequences of the polypeptide codes of the invention stored on a computer readable medium to reference sequences stored on a computer readable medium to identify homologies, motifs implicated in biological function, or structural motifs. The various sequence comparer programs identified elsewhere in this patent specification are particularly contemplated for use in this aspect of the invention.

FIG. 4 is a flow diagram illustrating one embodiment of a process 200 for comparing a new nucleotide or protein sequence with a database of sequences in order to determine the homology levels between the new sequence and the sequences in the database. The database of sequences can be a private database stored within the computer system 100, or a public database such as GENBANK, PIR OR SWISSPROT that is available through the Internet.

The process 200 begins at a start state 201 and then moves to a state 202 wherein the new sequence to be compared is stored to a memory in a computer system 100. As discussed above, the memory could be any type of memory, including RAM or an internal storage device.

The process 200 then moves to a state 204 wherein a database of sequences is opened for analysis and comparison. The process 200 then moves to a state 206 wherein the first sequence stored in the database is read into a memory on the computer. A comparison is then performed at a state 210 to determine if the first sequence is the same as the second sequence. It is important to note that this step is not limited to performing an exact comparison between the new sequence and the first sequence in the database. Well-known methods are known to those of skill in the art for comparing two nucleotide or protein sequences, even if they are not identical. For example, gaps can be introduced into one sequence in order to raise the homology level between the two tested sequences. The parameters that control whether gaps or other features are introduced into a sequence during comparison are normally entered by the user of the computer system.

Once a comparison of the two sequences has been performed at the state 210, a determination is made at a decision state 210 whether the two sequences are the same. Of course, the term “same” is not limited to sequences that are absolutely identical. Sequences that are within the homology parameters entered by the user will be marked as “same” in the process 200.

If a determination is made that the two sequences are the same, the process 200 moves to a state 214 wherein the name of the sequence from the database is displayed to the user. This state notifies the user that the sequence with the displayed name fulfills the homology constraints that were entered. Once the name of the stored sequence is displayed to the user, the process 200 moves to a decision state 218 wherein a determination is made whether more sequences exist in the database. If no more sequences exist in the database, then the process 200 terminates at an end state 220. However, if more sequences do exist in the database, then the process 200 moves to a state 224 wherein a pointer is moved to the next sequence in the database so that it can be compared to the new sequence. In this manner, the new sequence is aligned and compared with every sequence in the database.

It should be noted that if a determination had been made at the decision state 212 that the sequences were not homologous, then the process 200 would move immediately to the decision state 218 in order to determine if any other sequences were available in the database for comparison.

Accordingly, one aspect of the present invention is a computer system comprising a processor, a data storage device having stored thereon a nucleic acid code of the invention or a By polypeptide code of the invention, a data storage device having retrievably stored thereon reference nucleotide sequences or polypeptide sequences to be compared to the nucleic acid code of the invention or polypeptide code of the invention and a sequence comparer for conducting the comparison. The sequence comparer may indicate a homology level between the sequences compared or identify structural motifs in the nucleic acid code of the invention and polypeptide codes of the invention or it may identify structural motifs in sequences which are compared to these nucleic acid codes and polypeptide codes. In some embodiments, the data storage device may have stored thereon the sequences of at least 2, 5, 10, 15, 20, 25, 30, or 50 of the nucleic acid codes of the invention or polypeptide codes of the invention.

Another aspect of the present invention is a method for determining the level of homology between a nucleic acid code of the invention and a reference nucleotide sequence, comprising the steps of reading the nucleic acid code and the reference nucleotide sequence through the use of a computer program which determines homology levels and determining homology between the nucleic acid code and the reference nucleotide sequence with the computer program. The computer program may be any of a number of computer programs for determining homology levels, including those specifically enumerated herein, including BLAST2N with the default parameters or with any modified parameters. The method may be implemented using the computer systems described above. The method may also be performed by reading 2, 5, 10, 15, 20, 25, 30, or 50 of the above described nucleic acid codes of the invention through the use of the computer program and determining homology between the nucleic acid codes and reference nucleotide sequences.

FIG. 5 is a flow diagram illustrating one embodiment of a process 250 in a computer for determining whether two sequences are homologous. The process 250 begins at a start state 252 and then moves to a state 254 wherein a first sequence to be compared is stored to a memory. The second sequence to be compared is then stored to a memory at a state 256. The process 250 then moves to a state 260 wherein the first character in the first sequence is read and then to a state 262 wherein the first character of the second sequence is read. It should be understood that if the sequence is a nucleotide sequence, then the character would normally be either A, T, C, G or U. If the sequence is a protein sequence, then it should be in the single letter amino acid code so that the first and sequence sequences can be easily compared.

A determination is then made at a decision state 264 whether the two characters are the same. If they are the same, then the process 250 moves to a state 268 wherein the next characters in the first and second sequences are read. A determination is then made whether the next characters are the same. If they are, then the process 250 continues this loop until two characters are not the same. If a determination is made that the next two characters are not the same, the process 250 moves to a decision state 274 to determine whether there are any more characters either sequence to read.

If there aren't any more characters to read, then the process 250 moves to a state 276 wherein the level of homology between the first and second sequences is displayed to the user. The level of homology is determined by calculating the proportion of characters between the sequences that were the same out of the total number of sequences in the first sequence. Thus, if every character in a first 100 nucleotide sequence aligned with a every character in a second sequence, the homology level would be 100%.

Alternatively, the computer program may be a computer program which compares the nucleotide sequences of the nucleic acid codes of the present invention, to reference nucleotide sequences in order to determine whether the nucleic acid code of the invention differs from a reference nucleic acid sequence at one or more positions. Optionally such a program records the length and identity of inserted, deleted or substituted nucleotides with respect to the sequence of either the reference polynucleotide or the nucleic acid code of the invention. In one embodiment, the computer program may be a program which determines whether the nucleotide sequences of the nucleic acid codes of the invention contain one or more single nucleotide polymorphisms (SNP) with respect to a reference nucleotide sequence. These single nucleotide polymorphisms may each comprise a single base substitution, insertion, or deletion.

Another aspect of the present invention is a method for determining the level of homology between a polypeptide code of the invention and a reference polypeptide sequence, comprising the steps of reading the polypeptide code of the invention and the reference polypeptide sequence through use of a computer program which determines homology levels and determining homology between the polypeptide code and the reference polypeptide sequence using the computer program.

Accordingly, another aspect of the present invention is a method for determining whether a nucleic acid code of the invention differs at one or more nucleotides from a reference nucleotide sequence comprising the steps of reading the nucleic acid code and the reference nucleotide sequence through use of a computer program which identifies differences between nucleic acid sequences and identifying differences between the nucleic acid code and the reference nucleotide sequence with the computer program. In some embodiments, the computer program is a program which identifies single nucleotide polymorphisms The method may be implemented by the computer systems described above and the method illustrated in FIG. 5. The method may also be performed by reading at least 2, 5, 10, 15, 20, 25, 30, or 50 of the nucleic acid codes of the invention and the reference nucleotide sequences through the use of the computer program and identifying differences between the nucleic acid codes and the reference nucleotide sequences with the computer program.

In other embodiments the computer based system may further comprise an identifier for identifying features within the nucleotide sequences of the nucleic acid codes of the invention or the amino acid sequences of the polypeptide codes of the invention.

An “identifier” refers to one or more programs which identifies certain features within the above-described nucleotide sequences of the nucleic acid codes of the invention or the amino acid sequences of the polypeptide codes of the invention. In one embodiment, the identifier may comprise a program which identifies an open reading frame in the cDNAs codes of the invention.

FIG. 6 is a flow diagram illustrating one embodiment of an identifier process 300 for detecting the presence of a feature in a sequence. The process 300 begins at a start state 302 and then moves to a state 304 wherein a first sequence that is to be checked for features is stored to a memory 115 in the computer system 100. The process 300 then moves to a state 306 wherein a database of sequence features is opened. Such a database would include a list of each feature's attributes along with the name of the feature. For example, a feature name could be “Initiation Codon” and the attribute would be “ATG”. Another example would be the feature name “TTATAA Box” and the feature attribute would be “TATAA”. An example of such a database is produced by the University of Wisconsin Genetics Computer Group (Worldwide Web address: gcg.com).

Once the database of features is opened at the state 306, the process 300 moves to a state 308 wherein the first feature is read from the database. A comparison of the attribute of the first feature with the first sequence is then made at a state 310. A determination is then made at a decision state 316 whether the attribute of the feature was found in the first sequence. If the attribute was found, then the process 300 moves to a state 318 wherein the name of the found feature is displayed to the user.

The process 300 then moves to a decision state 320 wherein a determination is made whether move features exist in the database. If no more features do exist, then the process 300 terminates at an end state 324. However, if more features do exist in the database, then the process 300 reads the next sequence feature at a state 326 and loops back to the state 310 wherein the attribute of the next feature is compared against the first sequence.

It should be noted, that if the feature attribute is not found in the first sequence at the decision state 316, the process 300 moves directly to the decision state 320 in order to determine if any more features exist in the database.

In another embodiment, the identifier may comprise a molecular modeling program which determines the 3-dimensional structure of the polypeptides codes of the invention. In some embodiments, the molecular modeling program identifies target sequences that are most compatible with profiles representing the structural environments of the residues in known three-dimensional protein structures. (See, e.g., Eisenberg et al., U.S. Pat. No. 5,436,850 issued Jul. 25, 1995, the disclosure of which is incorporated herein by reference in its entirety). In another technique, the known three-dimensional structures of proteins in a given family are superimposed to define the structurally conserved regions in that family. This protein modeling technique also uses the known three-dimensional structure of a homologous protein to approximate the structure of the polypeptide codes of the invention. (See e.g., Srinivasan, et al., U.S. Pat. No. 5,557,535 issued Sep. 17, 1996, the disclosure of which is incorporated herein by reference in its entirety). Conventional homology modeling techniques have been used routinely to build models of proteases and antibodies. (Sowdhamini et al., 1997). Comparative approaches can also be used to develop three-dimensional protein models when the protein of interest has poor sequence identity to template proteins. In some cases, proteins fold into similar three-dimensional structures despite having very weak sequence identities. For example, the three-dimensional structures of a number of helical cytokines fold in similar three-dimensional topology in spite of weak sequence homology.

The recent development of threading methods now enables the identification of likely folding patterns in a number of situations where the structural relatedness between target and template(s) is not detectable at the sequence level. Hybrid methods, in which fold recognition is performed using Multiple Sequence Threading (MST), structural equivalencies are deduced from the threading output using a distance geometry program DRAGON to construct a low resolution model, and a full-atom representation is constructed using a molecular modeling package such as QUANTA.

According to this 3-step approach, candidate templates are first identified by using the novel fold recognition algorithm MST, which is capable of performing simultaneous threading of multiple aligned sequences onto one or more 3-D structures. In a second step, the structural equivalencies obtained from the MST output are converted into interresidue distance restraints and fed into the distance geometry program DRAGON, together with auxiliary information obtained from secondary structure predictions. The program combines the restraints in an unbiased manner and rapidly generates a large number of low resolution model confirmations. In a third step, these low resolution model confirmations are converted into full-atom models and subjected to energy minimization using the molecular modeling package QUANTA. (See e.g., Aszodi et al., 1997).

The results of the molecular modeling analysis may then be used in rational drug design techniques to identify agents which modulate the activity of the polypeptide codes of the invention.

Accordingly, another aspect of the present invention is a method of identifying a feature within the nucleic acid codes of the invention or the polypeptide codes of the invention comprising reading the nucleic acid code(s) or the polypeptide code(s) through the use of a computer program which identifies features therein and identifying features within the nucleic acid code(s) or polypeptide code(s) with the computer program. In one embodiment, computer program comprises a computer program which identifies open reading frames. In a further embodiment, the computer program identifies structural motifs in a polypeptide sequence. In another embodiment, the computer program comprises a molecular modeling program. The method may be performed by reading a single sequence or at least 2, 5, 10, 15, 20, 25, 30, or 50 of the nucleic acid codes of the invention or the polypeptide codes of the invention through the use of the computer program and identifying features within the nucleic acid codes or polypeptide codes with the computer program.

The nucleic acid codes of the invention or the polypeptide codes of the invention may be stored and manipulated in a variety of data processor programs in a variety of formats. For example, they may be stored as text in a word processing file, such as MicrosoftWORD or WORDPERFECT or as an ASCII file in a variety of database programs familiar to those of skill in the art, such as DB2, SYBASE, or ORACLE. In addition, many computer programs and databases may be used as sequence comparers, identifiers, or sources of reference nucleotide or polypeptide sequences to be compared to the nucleic acid codes of the invention or the polypeptide codes of the invention. The following list is intended not to limit the invention but to provide guidance to programs and databases which are useful with the nucleic acid codes of the invention or the polypeptide codes of the invention. The programs and databases which may be used include, but are not limited to: MacPattern (EMBL), DiscoveryBase (Molecular Applications Group), GeneMine (Molecular Applications Group), Look (Molecular Applications Group), MacLook (Molecular Applications Group), BLAST and BLAST2 (NCBI), BLASTN and BLASTX (Altschul et al, 1990), FASTA (Pearson and Lipman, 1988), FASTDB (Brutlag et al., 1990), Catalyst (Molecular Simulations Inc.), Catalyst/SHAPE (Molecular Simulations Inc.), Cerius².DBAccess (Molecular Simulations Inc.), HypoGen (Molecular Simulations Inc.), Insight II, (Molecular Simulations Inc.), Discover (Molecular Simulations Inc.), CHARMm (Molecular Simulations Inc.), Felix (Molecular Simulations Inc.), DelPhi, (Molecular Simulations Inc.), QuanteMM, (Molecular Simulations Inc.), Homology (Molecular Simulations Inc.), Modeler (Molecular Simulations Inc.), ISIS (Molecular Simulations Inc.), Quanta/Protein Design (Molecular Simulations Inc.), WebLab (Molecular Simulations Inc.), WebLab Diversity Explorer (Molecular Simulations Inc.), Gene Explorer (Molecular Simulations Inc.), SeqFold (Molecular Simulations Inc.), the EMBL/Swissprotein database, the MDL Available Chemicals Directory database, the MDL Drug Data Report data base, the Comprehensive Medicinal Chemistry database, Derwents's World Drug Index database, the BioByteMasterFile database, the Genbank database, and the Genseqn database. Many other programs and data bases would be apparent to one of skill in the art given the present disclosure.

Motifs which may be detected using the above programs include sequences encoding leucine zippers, helix-turn-helix motifs, glycosylation sites, ubiquitination sites, alpha helices, and beta sheets, signal sequences encoding signal peptides which direct the secretion of the encoded proteins, sequences implicated in transcription regulation such as homeoboxes, acidic stretches, enzymatic active sites, substrate binding sites, and enzymatic cleavage sites.

Throughout this application, various publications, patents and published patent applications are cited. The disclosures of these publications, patents and published patent specification referenced in this application are hereby incorporated by reference into the present disclosure to more fully describe the sate of the art to which this invention pertains.

EXAMPLES Example 1 Detection of PCTA-1 Biallelic Markers

DNA Extraction

Blood donors were from French Caucasian origin. They presented a sufficient diversity for being representative of a French heterogeneous population. The DNA from 100 unrelated and healthy individuals was extracted, pooled and tested for the detection of biallelic markers. The pool was constituted by mixing equivalent quantities of DNA from each individual.

30 ml of peripheral venous blood were taken from each donor in the presence of EDTA. Cells (pellet) were collected after centrifugation for 10 minutes at 2000 rpm. Red cells were lysed by a lysis solution (50 ml final volume: 10 mM Tris pH7.6; 5 mM MgCl₂; 10 mM NaCl). The solution was centrifuged (10 minutes, 2000 rpm) as many times as necessary to eliminate the residual red cells present in the supernatant, after resuspension of the pellet in the lysis solution.

The pellet of white cells was lysed overnight at 42° C. with 3.7 ml of lysis solution composed of:

3 ml TE 10-2 (Tris-HCl 10 mM, EDTA 2 mM)/NaCl 0.4 M

200 μl SDS 10%

500 μl K-proteinase (2 mg K-proteinase in TE 10-2/NaCl 0.4 M).

For the extraction of proteins, 1 ml saturated NaCl (6M) (1/3.5 v/v) was added. After vigorous agitation, the solution was centrifuged for 20 minutes at 10000 rpm.

For the precipitation of DNA, 2 to 3 volumes of 100% ethanol were added to the previous supernatant, and the solution was centrifuged for 30 minutes at 2000 rpm. The DNA solution was rinsed three times with 70% ethanol to eliminate salts, and centrifuged for 20 minutes at 2000 rpm. The pellet was dried at 37° C., and resuspended in 1 ml TE 10- 1 or 1 ml water. The DNA concentration was evaluated by measuring the OD at 260 nm (1 unit OD=50 μg/ml DNA).

To determine the presence of proteins in the DNA solution, the OD 260/OD 280 ratio was determined. Only DNA preparations having a OD 260/OD 280 ratio between 1.8 and 2 were used in the subsequent examples described below.

Example 2

Detection of the Biallelic Markers

Amplification of Genomic DNA by PCR

The amplification of specific genomic sequences of the DNA samples of example 1 was carried out on the pool of DNA obtained previously. In addition, 10 individual samples were similarly amplified.

Final volume   25 μl DNA   2 ng/μl MgCl₂   2 mM dNTP (each)  200 μM primer (each)  2.9 ng/μl Ampli Taq Gold DNA polymerase 0.05 unit/μl PCR buffer (10× = 0.1M TrisHCl pH 8.3 0.5M KCl 1×

Each pair of primers was designed using the sequence information of our total genomic sequence (SEQ ID No 1) and the OSP software (Hillier& Green, 1991). These primers had about 20 nucleotides in length and their respective sequences are disclosed in Table 1 and had the sequences disclosed in Table 1 in the columns labeled “Position range of amplification primer in SEQ ID No 1” and “Complementary position range of amplification primer in SEQ ID No 1”.

The primers contained a common oligonucleotide tail upstream of the specific bases targeted for amplification which was useful for sequencing.

Primers from the columns labeled “Position range of amplification primer in SEQ ID No 1,” contain the following additional PU 5′ sequence: TGTAAAACGACGGCCAGT; and primers from the columns labeled “Complementary position range of amplification primer in SEQ ID No 1,” contain the following RP 5′ sequence: CAGGAAACAGCTATGACC. The primer containing the additional PU 5′ sequence is listed in SEQ ID No 10. The primer containing the additional RP 5′ sequence is listed in SEQ ID No 11.

The synthesis of these primers was performed following the phosphoramidite method, on a GENSET UFPS 24.1 synthesizer.

DNA amplification was performed on a Genius II thermocycler. After heating at 94° C. for 10 min, 40 cycles were performed. Each cycle comprised: 30 sec at 94° C., 55° C. for 1 min, and 30 sec at 72° C. For final elongation, 7 min at 72° C. end the amplification. The quantities of the amplification products obtained were determined on 96-well microtiter plates, using a fluorometer and Picogreen as intercalant agent (Molecular Probes).

TABLE 1 Complementary Position range Position range of position range of of the amplicon Primer amplification primer primer amplification primer Amplicon in SEQ ID 1 name in SEQ ID No 1 name in SEQ ID No 1 99-1601 1 506 B1  1 18 C1  486 506 99-13801 2607 3054 B2  2607 2627 C2  3035 3054 99-13806 11883 12331 B3  11883 11902 C3  12313 12331 99-13799 12379 12909 B4  12379 12399 C4  12889 12909 99-13798 17442 17887 B5  17442 17462 C5  17868 17887 99-1602 21881 22506 B6  21881 21899 C6  22487 22506 99-13794 28669 29149 B7  28669 28689 C7  29131 29149 99-13812 30941 31457 B8  30941 30961 C8  31437 31457 99-13805 31560 32075 B9  31560 31579 C9  32057 32075 99-1587 34515 34909 B10 34515 34535 C10 34890 34909 99-1582 45325 46018 B11 45325 45343 C11 46000 46018 99-1585 49765 50310 B12 49765 49784 C12 50291 50310 99-1607 54726 55325 B13 54726 54746 C13 55307 55325 99-1577 64135 64536 B14 64135 64153 C14 64518 64536 99-1591 65202 65834 B15 65202 65219 C15 65815 65834 99-1572 66653 67295 B16 66653 66671 C16 67275 67295 5-169 67627 68043 B17 67627 67646 C17 68024 68043 5-264 67246 67696 B18 67246 67263 C18 67678 67696 5-170 67977 68424 B19 67977 67994 C19 68406 68424 5-171 68322 68742 B20 68322 68340 C20 68725 68742 5-1 70507 70928 B21 70507 70524 C21 70909 70928 99-1578 79940 80575 B22 79940 79957 C22 80557 80575 99-1605 82057 82504 B23 82057 82077 C23 82484 82504 5-2 82058 82492 B24 82058 82077 C24 82473 82492 5-3 83561 83982 B25 83561 83578 C25 83965 83982 5-4 83597 84017 B26 83597 83616 C26 83999 84017 5-260 83793 84167 B27 83793 83812 C27 84148 84167 5-9 85153 85576 B28 85153 85170 C28 85559 85576 5-5 86239 86539 B29 86239 86257 C29 86519 86539 5-202 87619 88050 B30 87619 87638 C30 88033 88050 5-7 88104 88536 B31 88104 88122 C31 88519 88536 5-181 89338 89758 B32 89338 89357 C32 89739 89758 5-10 92722 93142 B33 92722 92741 C33 93124 93142 5-11 93090 93509 B34 93090 93108 C34 93490 93509 5-12 93460 93881 B35 93460 93478 C35 93862 93881 5-13 93759 94192 B36 93759 93776 C36 94175 94192 5-14 94127 94554 B37 94127 94144 C37 94535 94554 5-15 94504 94921 B38 94504 94521 C38 94904 94921 5-16 94833 95251 B39 94833 94850 C39 95232 95251 5-17 95124 95561 B40 95124 95142 C40 95542 95561 5-18 95290 95708 B41 95290 95308 C41 95689 95708 5-300 95533 95952 B42 95533 95551 C42 95934 95952 5-262 96097 96591 B43 96097 96115 C43 96574 96591 5-263 96548 97001 B44 96548 96565 C44 96982 97001 5-265 96901 97309 B45 96901 96918 C45 97292 97309 99-7183 102156 102604 B46 102156 102176 C46 102584 102604 99-7207 105570 106074 B47 105570 105588 C47 106056 106074

Example 3

Detection of the Biallelic Markers

Sequencing of Amplified Genomic DNA and Identification of Polymorphisms

The sequencing of the amplified DNA obtained in example 2 was carried out on ABI 377 sequencers. The sequences of the amplification products were determined using automated dideoxy terminator sequencing reactions with a dye terminator cycle sequencing protocol. The products of the sequencing reactions were run on sequencing gels and the sequences were determined using gel image analysis (ABI Prism DNA Sequencing Analysis software (2.1.2 version)).

The sequence data were further evaluated using the above mentioned polymorphism analysis software designed to detect the presence of biallelic markers among the pooled amplified fragments. The polymorphism search was based on the presence of superimposed peaks in the electrophoresis pattern resulting from different bases occurring at the same position as described previously.

47 fragments of amplification were analyzed. In these segments, 125 markers were detected. The localization of the biallelic markers was as shown in Table 2. Table 3 comprises the polynucleotides defining the PCTA-1-related biallelic markers. They could be used as probes and their sequence are disclosed in Table 3 in “Position range of probes in SEQ ID No 1”.

TABLE 2 Localization Polymorphism Marker in PCTA-1 (frequency %) BM position in SEQ ID Amplicon BM Name gene all1 all2 No 1 No 2 No 3 No 4 99-1601 A1  99-1601-278 5′regulatory A C 278 99-1601 A2  99-1601-402 5′regulatory A (66) T 402 99-1601 A3  99-1601-472 5′regulatory A T 472 99-13801 A4  99-13801-100 5′regulatory T C 2955 99-13806 A5  99-13806-166 5′regulatory G A 12167 99-13799 A6  99-13799-376 5′regulatory T G 12536 99-13798 A7  99-13798-297 5′regulatory T C 17593 99-13798 A8  99-13798-284 5′regulatory T C 17606 99-1602 A9  99-1602-200 5′regulatory C G 22079 99-13794 A10 99-13794-186 5′regulatory T C 28964 99-13794 A11 99-13794-147 5′regulatory C G 29003 99-13812 A12 99-13812-384 5′regulatory T C 31077 99-13805 A13 99-13805-313 5′regulatory T C 31766 99-1587 A14 99-1587-281 5′regulatory A G 34791 99-1582 A15 99-1582-430 5′regulatory C T 45751 99-1585 A16 99-1585-465 5′regulatory T C 49847 99-1585 A17 99-1585-457 5′regulatory T C 49855 99-1585 A18 99-1585-426 5′regulatory G A 49886 99-1585 A19 99-1585-412 5′regulatory G A 49900 99-1585 A20 99-1585-406 5′regulatory C A 49906 99-1585 A21 99-1585-391 5′regulatory C A 49921 99-1585 A22 99-1585-373 5′regulatory G A 49939 99-1585 A23 99-1585-55 5′regulatory C A 50256 99-1607 A24 99-1607-373 5′regulatory T C 54955 99-1577 A25 99-1577-105 5′regulatory A (54) G 64239 99-1591 A26 99-1591-235 5′regulatory A G 65436 99-1591 A27 99-1591-295 5′regulatory G T 65496 99-1572 A28 99-1572-315 Promoter C T 66967 99-1572 A29 99-1572-335 Promoter A G 66987 99-1572 A30 99-1572-440 Promoter C (32) T 67092 99-1572 A31 99-1572-477 Promoter A T 67129 99-1572 A32 99-1572-578 Promoter C T 67229 5-264 A33 5-264-188 Promoter A G 67433 5-169 A34 5-169-97 Promoter G (18) C 67723 5-169 A35 5-169-208 Promoter A (<1) G 67834 5-169 A36 5-169-331 Promoter C (99) T 67955 5-170 A37 5-170-238 Promoter A G 68213 5-170 A38 5-170-288 Promoter A (1) C 68263 5-170 A39 5-170-400 Promoter G C 68375 5-171 A40 5-171-156 Promoter G T 68477 5-171 A41 5-171-204 Promoter C (30) T 68525 5-171 A42 5-171-273 Promoter A G 68594 5-171 A43 5-171-289 Promoter C T 68610 5-1 A44 5-1-60 Intron 0 C (1) T 70566 5-1 A45 5-1-222 Exon 1 A G 70728 176 176 176 99-1578 A46 99-1578-99 Intron 1 G T 80038 99-1578 A47 99-1578-179 Intron 1 A T 80118 99-1578 A48 99-1578-231 Intron 1 Ins 80170 AC 99-1578 A49 99-1578-245 Intron 1 del AT 80183 99-1578 A50 99-1578-496 Intron 1 C T 80435 5-2 A51 5-2-30 Intron 1 Ins 82090 CAG 5-2 A52 5-2-109 Intron 1 G T 82165 5-2 A53 5-2-113 Intron 1 Del 82169 GTTT 5-2 A54 5-2-162 Exon 2 A (67) T 82218 253 253 253 5-2 A55 5-2-178 Exon 2 C (67) T 82234 269 269 269 5-2 A56 5-2-213 Exon 2 C (33) T 82268 303 303 303 99-1605 A57 99-1605-112 Intron 2 T (67) C 82393 5-3 A58 5-3-27 Intron 2 A G 83587 5-3 A59 5-3-83 Exon 3 C (39) T 83643 362 362 362 5-3 A60 5-3-84 Exon 3 A (29) G 83644 363 363 363 5-3 A61 5-3-248 Exon 3 A G 83808 527 527 527 5-3 A62 5-3-321 Intron 3 G T 83881 5-3 A63 5-3-324 Intron 3 C T 83884 5-4 A64 5-4-313 Intron 3 A G 83909 5-3 A65 5-3-377 Intron 3 ins 83937 TTTG 5-4 A66 5-4-351 Intron 3 C T 83947 5-4 A67 5-4-386 Intron 3 A G 83982 5-4 A68 5-4-392 Intron 3 GGG TA 83988 5-260 A69 5-260-255 Intron 3 C T 84047 5-260 A70 5-260-300 Intron 3 C T 84092 5-260 A71 5-260-353 Intron 3 C T 84145 5-9 A72 5-9-50 Intron 3 C T 85202 5-5 A73 5-5-21 Intron 4 A G 86259 5-5 A74 5-5-85 Intron 4 TATA ACAG 86323 AAAT GTTA ATT TATA 5-202 A75 5-202-95 Exon 6bis G T (<1) 87713 810 5-202 A76 5-202-117 Exon 6bis A (<1) T 87735 832 5-202 A77 5-202-169 Intron 6bis A C 87787 5-202 A78 5-202-188 Intron 6bis A G 87806 5-202 A79 5-202-242 Intron 6bis A G 87860 5-202 A80 5-202-284 Intron 6bis C T 87902 5-202 A81 5-202-362 Intron 6bis del CC 87980 5-202 A82 5-202-394 Intron 6bis C T 88012 5-7 A83 5-7-113 Intron 6bis C T 88215 5-7 A84 5-7-181 Intron 6bis G C 88283 5-7 A85 5-7-195 Exon 7 G (25) C 88297 749 875 749 5-7 A86 5-7-340 Intron 7 C T 88442 5-7 A87 5-7-369 Intron 7 A T 88471 5-7 A88 5-7-378 Intron 7 C T 88480 5-181 A89 5-181-57 Intron 7 A G 89394 5-181 A90 5-181-127 Intron 7 C T 89464 5-181 A91 5-181-134 Intron 7 C T 89471 5-181 A92 5-181-321 Intron 8 A C 89658 5-10 A93 5-10-39 Exon 9 C T 92760 1013 1139 1013 exon 9bis 5-10 A94 5-10-302 Exon 9 A G 93023 1276 1402 Intron 9bis 5-10 A95 5-10-334 Exon 9 A C 93055 1308 1434 Intron 9bis 5-11 A96 5-11-158 Exon 9 A (22) G 93247 1500 1626 Intron 9bis 5-11 A97 5-11-230 Exon 9 G T 93319 1572 1698 Intron 9bis 5-11 A98 5-11-234 Exon9 C T 93323 1576 1702 Intron 9bis 5-11 A99 5-11-299 Exon9 A T 93388 1641 1767 Intron 9bis 5-11  A100 5-11-304 Exon9 A C 93393 1646 1772 Intron 9bis 5-11  A101 5-11-329 Exon 9 C T 93418 1671 1797 Intron 9bis 5-12  A102 5-12-56 Exon 9 ins 93515 1768 1894 Intron 9bis CTTT 5-12  A103 5-12-267 Exon 9 A C 93726 1979 2105 Intron 9bis 5-13  A104 5-13-145 Exon 9 C T 93903 2156 2282 Intron 9bis 5-14  A105 5-14-44 Exon 9 C T 94170 2423 2549 Intron 9bis 5-14  A106 5-14-93 Exon 9 A T 94218 2471 2597 Intron 9bis 5-14  A107 5-14-144 Exon 9 ins T 94269 2522 2648 Intron 9bis 5-14  A108 5-14-165 Exon 9 C T 94290 2543 2669 Intron 9bis 5-14  A109 5-14-297 Exon 9 A C 94422 2675 2801 Intron 9bis 5-14  A110 5-14-307 Exon 9 G T 94432 2685 2811 Intron 9bis 5-15  A111 5-15-219 Exon 9 A T 94720 2973 3099 Intron 9bis 5-16  A112 5-16-157 Exon 9 A G 94989 3242 3368 Intron 9bis 5-17  A113 5-17-140 Exon 9 A G 95261 3514 3640 Intron 9bis 5-18  A114 5-18-51 Exon 9 G T 95340 3593 3719 Intron 9bis 5-18  A115 5-18-208 Exon 9 A C 95497 3750 3876 Intron 9bis 5-300  A116 5-300-238 Exon 9 C T 95770 4023 4149 Intron 9bis 5-300  A117 5-300-287 Exon 9 A G 95819 4072 4198 Intron 9bis 5-262  A118 5-262-49 Exon 9 ins C 96145 4398 4524 1461 Exon 9ter 5-262  A119 5-262-85 Exon 9 C T 96181 4434 4560 1497 Exon 9ter 5-262  A120 5-262-254 Exon 9 C T 96350 4603 4729 1666 Exon 9ter 5-263  A121 5-263-404 Exon 9 C T 96951 5204 5330 2267 Exon 9ter 5-265  A122 5-265-244 Exon 9 A G 97144 5397 5523 2460 Exon 9ter 5-265  A123 5-265-376 3′regulatory A G 97276 99-7183  A124 99-7183-338 3′regulatory C T 102267 99-7207  A125 99-7207-138 3′regulatory A G 105937

BM refers to “biallelic marker”. A111 and a112 refer respectively to allele 1 and allele 2 of the biallelic marker. “Frequency%” refers to the frequency of the allele in percentage in control population. Frequencies corresponded to a population of random blood donors of French Caucasian origin.

TABLE 3 Position range of probes in SEQ BM Marker Name ID No 1 Probes A1 99-1601-278 255 301 P1 A2 99-1601-402 379 425 P2 A3 99-1601-472 449 495 P3 A4 99-13801-100 2932 2978 P4 A5 99-13806-166 12144 12190 P5 A6 99-13799-376 12513 12559 P6 A7 99-13798-297 17570 17616 P7 A8 99-13798-284 17583 17629 P8 A9 99-1602-200 22056 22102 P9 A10 99-13794-186 28941 28987 P10 A11 99-13794-147 28980 29026 P11 A12 99-13812-384 31054 31100 P12 A13 99-13805-313 31743 31789 P13 A14 99-1587-281 34768 34814 P14 A15 99-1582-430 45728 45774 P15 A16 99-1585-465 49824 49870 P16 A17 99-1585-457 49832 49878 P17 A18 99-1585-426 49863 49909 P18 A19 99-1585-412 49877 49923 P19 A20 99-1585-406 49883 49929 P20 A21 99-1585-391 49898 49944 P21 A22 99-1585-373 49916 49962 P22 A23 99-1585-55 50233 50279 P23 A24 99-1607-373 54932 54978 P24 A25 99-1577-105 64216 64262 P25 A26 99-1591-235 65413 65459 P26 A27 99-1591-295 65473 65519 P27 A28 99-1572-315 66944 66990 P28 A29 99-1572-335 66964 67010 P29 A30 99-1572-440 67069 67115 P30 A31 99-1572-477 67106 67152 P31 A32 99-1572-578 67206 67252 P32 A33 5-264-188 67410 67456 P33 A34 5-169-97 67700 67746 P34 A35 5-169-208 67811 67857 P35 A36 5-169-331 67932 67978 P36 A37 5-170-238 68190 68236 P37 A38 5-170-288 68240 68286 P38 A39 5-170-400 68352 68398 P39 A40 5-171-156 68454 68500 P40 A41 5-171-204 68502 68548 P41 A42 5-171-273 68571 68617 P42 A43 5-171-289 68587 68633 P43 A44 5-1-60 70543 70589 P44 A45 5-1-222 70705 70751 P45 A46 99-1578-99 80015 80061 P46 A47 99-1578-179 80095 80141 P47 A48 99-1578-231 80147 80193 P48 A49 99-1578-245 80160 80206 P49 A50 99-1578-496 80412 80458 P50 A51 5-2-30 82067 82113 P51 A52 5-2-109 82142 82188 P52 A53 5-2-113 82146 82192 P53 A54 5-2-162 82195 82241 P54 A55 5-2-178 82211 82257 P55 A56 5-2-213 82245 82291 P56 A57 99-1605-112 82370 82416 P57 A58 5-3-27 83564 83610 P58 A59 5-3-83 83620 83666 P59 A60 5-3-84 83621 83667 P60 A61 5-3-248 83785 83831 P61 A62 5-3-321 83858 83904 P62 A63 5-3-324 83861 83907 P63 A64 5-4-313 83886 83932 P64 A65 5-3-377 83914 83960 P65 A66 5-4-351 83924 83970 P66 A67 5-4-386 83959 84005 P67 A68 5-4-392 83965 84011 P68 A69 5-260-255 84024 84070 P69 A70 5-260-300 84069 84115 P70 A71 5-260-353 84122 84168 P71 A72 5-9-50 85179 85225 P72 A73 5-5-21 86236 86282 P73 A74 5-5-85 86300 86346 P74 A75 5-202-95 87690 87736 P75 A76 5-202-117 87712 87758 P76 A77 5-202-169 87764 87810 P77 A78 5-202-188 87783 87829 P78 A79 5-202-242 87837 87883 P79 A80 5-202-284 87879 87925 P80 A81 5-202-362 87957 88003 P81 A82 5-202-394 87989 88035 P82 A83 5-7-113 88192 88238 P83 A84 5-7-181 88260 88306 P84 A85 5-7-195 88274 88320 P85 A86 5-7-340 88419 88465 P86 A87 5-7-369 88448 88494 P87 A88 5-7-378 88457 88503 P88 A89 5-181-57 89371 89417 P89 A90 5-181-127 89441 89487 P90 A91 5-181-134 89448 89494 P91 A92 5-181-321 89635 89681 P92 A93 5-10-39 92737 92783 P93 A94 5-10-302 93000 93046 P94 A95 5-10-334 93032 93078 P95 A96 5-11-158 93224 93270 P96 A97 5-11-230 93296 93342 P97 A98 5-11-234 93300 93346 P98 A99 5-11-299 93365 93411 P99 A100 5-11-304 93370 93416 P100 A101 5-11-329 93395 93441 P101 A102 5-12-56 93492 93538 P102 A103 5-12-267 93703 93749 P103 A104 5-13-145 93880 93926 P104 A105 5-14-44 94147 94193 P105 A106 5-14-93 94195 94241 P106 A107 5-14-144 94246 94292 P107 A108 5-14-165 94267 94313 P108 A109 5-14-297 94399 94445 P109 A110 5-14-307 94409 94455 P110 A111 5-15-219 94697 94743 P111 A112 5-16-157 94966 95012 P112 A113 5-17-140 95238 95284 P113 A114 5-18-51 95317 95363 P114 A115 5-18-208 95474 95520 P115 A116 5-300-238 95747 95793 P116 A117 5-300-287 95796 95842 P117 A118 5-262-49 96122 96168 P118 A119 5-262-85 96158 96204 P119 A120 5-262-254 96327 96373 P120 A121 5-263-404 96928 96974 P121 A122 5-265-244 97121 97167 P122 A123 5-265-376 97253 97299 P123 A124 99-7183-338 102244 102290 P124 A125 99-7207-138 105914 105960 P125

Example 4 Validation of the Polymorphisms through Microseguencing

The biallelic markers identified in example 3 were further confirmed and their respective frequencies were determined through microsequencing. Microsequencing was carried out for each individual DNA sample described in Example 1.

Amplification from genomic DNA of individuals was performed by PCR as described above for the detection of the biallelic markers with the same set of PCR primers (Table 1).

The preferred primers used in microsequencing had about 19 nucleotides in length and hybridized just upstream of the considered polymorphic base. Their sequences are disclosed in Table 4 in columns labeled “Position range of microsequencing primer mis. 1 in SEQ ID No 1” and “Complementary position range of microsequencing primer mis. 2 in SEQ ID No 1”.

Mis 1 and Mis 2 respectively refer to microsequencing primers which hybridized with the non-coding strand of the PCTA-1 gene or with the coding strand of the PCTA-1 gene.

The microsequencing reaction was performed as follows:

10 μl of PCR products were added to 20 μl of microsequencing reaction mixture containing: 10 pmol microsequencing oligonucleotide (crude synthesis, 5 OD), 1 U Thermosequenase (Amersham E79000G), 1.25 μl Thermosequenase buffer (260 mM Tris HCl pH 9.5, 65 mM MgCl₂), and the appropriate fluorescent ddNTPs complementary to the nucleotides at the polymorphic site corresponding to the polymorphic bases (11.25 nM TAMRA-ddTTP; 16.25 nM ROX-ddCTP; 1.675 nM REG-ddATP; 1.25 nM RHO-ddGTP; Perkin Elmer, Dye Terminator Set 401095). After 4 minutes at 94° C., 20 PCR cycles of 15 sec at 55° C., 5 sec at 72° C., and 10 sec at 94° C. were carried out in a thermocycler. After amplification, the unincorporated dye terminators were removed by ethanol precipitation. After discarding the supernatants, the microplate was evaporated to dryness under reduced pressure (Speed Vac); samples were resuspended in 2.5 μl formamide EDTA loading buffer and heated for 2 min at 95° C. 0.8 μl microsequencing reaction were loaded on a 10% (19:1) polyacrylamide sequencing gel. The data were collected by an ABI PRISM 377 DNA sequencer and processed using the GENESCAN software (Perkin Elmer).

Following gel analysis, data were automatically processed with software that allows the determination of the alleles of biallelic markers present in each amplified fragment.

The software evaluates such factors as whether the intensities of the signals resulting from the above microsequencing procedures are weak, normal, or saturated, or whether the signals are ambiguous. In addition, the software identifies significant peaks (according to shape and height criteria). Among the significant peaks, peaks corresponding to the targeted site are identified based on their position. When two significant peaks are detected for the same position, each sample is categorized classification as homozygous or heterozygous type based on the height ratio.

TABLE 4 Complementary Position range of position range of microsequencing microsequencing primer mis. 1 in primer mis. 2 in Marker Name BM Mis. 1 SEQ ID No 1 Mis. 2 SEQ ID No 1 99-1601-278 A1  D1  258 277 E1  279 298 99-1601-402 A2  D2  382 401 E2  403 422 99-1601-472 A3  D3  452 471 E3  473 492 99-13801-100 A4  D4  2935 2954 E4  2956 2975 99-13806-166 A5  D5  12147 12166 E5  12168 12187 99-13799-376 A6  D6  12516 12535 E6  12537 12556 99-13798-297 A7  D7  17573 17592 E7  17594 17613 99-13798-284 A8  D8  17586 17605 E8  17607 17626 99-1602-200 A9  D9  22059 22078 E9  22080 22099 99-13794-186 A10 D10 28944 28963 E10 28965 28984 99-13794-147 A11 D11 28983 29002 E11 29004 29023 99-13812-384 A12 D12 31057 31076 E12 31078 31097 99-13805-313 A13 D13 31746 31765 E13 31767 31786 99-1587-281 A14 D14 34771 34790 E14 34792 34811 99-1582-430 A15 D15 45731 45750 E15 45752 45771 99-1585-465 A16 D16 49827 49846 E16 49848 49867 99-1585-457 A17 D17 49835 49854 E17 49856 49875 99-1585-426 A18 D18 49866 49885 E18 49887 49906 99-1585-412 A19 D19 49880 49899 E19 49901 49920 99-1585-406 A20 D20 49886 49905 E20 49907 49926 99-1585-391 A21 D21 49901 49920 E21 49922 49941 99-1585-373 A22 D22 49919 49938 E22 49940 49959 99-1585-55 A23 D23 50236 50255 E23 50257 50276 99-1607-373 A24 D24 54935 54954 E24 54956 54975 99-1577-105 A25 D25 64219 64238 E25 64240 64259 99-1591-235 A26 D26 65416 65435 E26 65437 65456 99-1591-295 A27 D27 65476 65495 E27 65497 65516 99-1572-315 A28 D28 66947 66966 E28 66968 66987 99-1572-335 A29 D29 66967 66986 E29 66988 67007 99-1572-440 A30 D30 67072 67091 E30 67093 67112 99-1572-477 A31 D31 67109 67128 E31 67130 67149 99-1572-578 A32 D32 67209 67228 E32 67230 67249 5-264-188 A33 D33 67413 67432 E33 67434 67453 5-169-97 A34 D34 67703 67722 E34 67724 67743 5-169-208 A35 D35 67814 67833 E35 67835 67854 5-169-331 A36 D36 67935 67954 E36 67956 67975 5-170-238 A37 D37 68193 68212 E37 68214 68233 5-170-288 A38 D38 68243 68262 E38 68264 68283 5-170-400 A39 D39 68355 68374 E39 68376 68395 5-171-156 A40 D40 68457 68476 E40 68478 68497 5-171-204 A41 D41 68505 68524 E41 68526 68545 5-171-273 A42 D42 68574 68593 E42 68595 68614 5-171-289 A43 D43 68590 68609 E43 68611 68630 5-1-60 A44 D44 70546 70565 E44 70567 70586 5-1-222 A45 D45 70708 70727 E45 70729 70748 99-1578-99 A46 D46 80018 80037 E46 80039 80058 99-1578-179 A47 D47 80098 80117 E47 80119 80138 99-1578-231 A48 D48 80150 80169 E48 80171 80190 99-1578-245 A49 D49 80163 80182 E49 80184 80203 99-1578-496 A50 D50 80415 80434 E50 80436 80455 5-2-30 A51 D51 82070 82089 E51 82091 82110 5-2-109 A52 D52 82145 82164 E52 82166 82185 5-2-113 A53 D53 82149 82168 E53 82170 82189 5-2-162 A54 D54 82198 82217 E54 82219 82238 5-2-178 A55 D55 82214 82233 E55 82235 82254 5-2-213 A56 D56 82248 82267 E56 82269 82288 99-1605-112 A57 D57 82373 82392 E57 82394 82413 5-3-27 A58 D58 83567 83586 E58 83588 83607 5-3-83 A59 D59 83623 83642 E59 83644 83663 5-3-84 A60 D60 83624 83643 E60 83645 83664 5-3-248 A61 D61 83788 83807 E61 83809 83828 5-3-321 A62 D62 83861 83880 E62 83882 83901 5-3-324 A63 D63 83864 83883 E63 83885 83904 5-4-313 A64 D64 83889 83908 E64 83910 83929 5-3-377 A65 D65 83917 83936 E65 83938 83957 5-4-351 A66 D66 83927 83946 E66 83948 83967 5-4-386 A67 D67 83962 83981 E67 83983 84002 5-4-392 A68 D68 83968 83987 E68 83989 84008 5-260-255 A69 D69 84027 84046 E69 84048 84067 5-260-300 A70 D70 84072 84091 E70 84093 84112 5-260-353 A71 D71 84125 84144 E71 84146 84165 5-9-50 A72 D72 85182 85201 E72 85203 85222 5-5-21 A73 D73 86239 86258 E73 86260 86279 5-5-85 A74 D74 86303 86322 E74 86324 86343 5-202-95 A75 D75 87693 87712 E75 87714 87733 5-202-117 A76 D76 87715 87734 E76 87736 87755 5-202-169 A77 D77 87767 87786 E77 87788 87807 5-202-188 A78 D78 87786 87805 E78 87807 87826 5-202-242 A79 D79 87840 87859 E79 87861 87880 5-202-284 A80 D80 87882 87901 E80 87903 87922 5-202-362 A81 D81 87960 87979 E81 87981 88000 5-202-394 A82 D82 87992 88011 E82 88013 88032 5-7-113 A83 D83 88195 88214 E83 88216 88235 5-7-181 A84 D84 88263 88282 E84 88284 88303 5-7-195 A85 D85 88277 88296 E85 88298 88317 5-7-340 A86 D86 88422 88441 E86 88443 88462 5-7-369 A87 D87 88451 88470 E87 88472 88491 5-7-378 A88 D88 88460 88479 E88 88481 88500 5-181-57 A89 D89 89374 89393 E89 89395 89414 5-181-127 A90 D90 89444 89463 E90 89465 89484 5-181-134 A91 D91 89451 89470 E91 89472 89491 5-181-321 A92 D92 89638 89657 E92 89659 89678 5-10-39 A93 D93 92740 92759 E93 92761 92780 5-10-302 A94 D94 93003 93022 E94 93024 93043 5-10-334 A95 D95 93035 93054 E95 93056 93075 5-11-158 A96 D96 93227 93246 E96 93248 93267 5-11-230 A97 D97 93299 93318 E97 93320 93339 5-11-234 A98 D98 93303 93322 E98 93324 93343 5-11-299 A99 D99 93368 93387 E99 93389 93408 5-11-304  A100  D100 93373 93392  E100 93394 93413 5-11-329  A101  D101 93398 93417  E101 93419 93438 5-12-56  A102  D102 93495 93514  E102 93516 93535 5-12-267  A103  D103 93706 93725  E103 93727 93746 5-13-145  A104  D104 93883 93902  E104 93904 93923 5-14-44  A105  D105 94150 94169  E105 94171 94190 5-14-93  A106  D106 94198 94217  E106 94219 94238 5-14-144  A107  D107 94249 94268  E107 94270 94289 5-14-165  A108  D108 94270 94289  E108 94291 94310 5-14-297  A109  D109 94402 94421  E109 94423 94442 5-14-307  A110  D110 94412 94431  E110 94433 94452 5-15-219  A111  D111 94700 94719  E111 94721 94740 5-16-157  A112  D112 94969 94988  E112 94990 95009 5-17-140  A113  D113 95241 95260  E113 95262 95281 5-18-51  A114  D114 95320 95339  E114 95341 95360 5-18-208  A115  D115 95477 95496  E115 95498 95517 5-300-238  A116  D116 95750 95769  E116 95771 95790 5-300-287  A117  D117 95799 95818  E117 95820 95839 5-262-49  A118  D118 96125 96144  E118 96146 96165 5-262-85  A119  D119 96161 96180  E119 96182 96201 5-262-254  A120  D120 96330 96349  E120 96351 96370 5-263-404  A121  D121 96931 96950  E121 96952 96971 5-265-244  A122  D122 97124 97143  E122 97145 97164 5-265-376  A123  D123 97256 97275  E123 97277 97296 99-7183-338  A124  D124 102247 102266  E124 102268 102287 99-7207-138  A125  D125 105917 105936  E125 105938 105957

Example 5 Association Study between Prostate Cancer and the Biallelic Markers of the PCTA-1 Gene

Collection of DNA Samples from Affected and Non-Affected Individuals

Affected Population:

The positive trait followed in this association study was prostate cancer. Prostate cancer patients were recruited according to a combination of clinical, histological and biological inclusion criteria. Clinical criteria can include rectal examination and prostate biopsies. Biological criteria can include PSA assays. The affected individuals were recorded as familial forms when at least two persons affected by prostate cancer have been diagnosed in the family. Remaining cases were classified as non-familial informative cases (at least two sibs of the case both aged over 50 years old are unaffected), or non-familial uniformative cases (no information about sibs over 50 years old is available). All affected individuals included in the statistical analysis of this patent were unrelated. Cases were also separated following the criteria of diagnosis age: early onset prostate cancer (under 65 years old) and late onset prostate cancer (65 years old or more).

Unaffected Population:

Control individuals included in this study were checked for both the absence of all clinical and biological criteria defining the presence or the risk of prostate cancer (PSA<4) (WO 96/21042), and for their age (aged 65 years old or more). All unaffected individuals included in the statistical analysis of this patent were unrelated.

The affected group was composed by 491 unrelated individuals, comprising:

197 familial cases among which 91 individuals were under 65 years old and 106 individuals were 65 years old or more; and

294 sporadic cases.

The unaffected group contained 313 individuals which were 65 years or older.

As used herein, the term “early onset cancer” refers to a cancer in which the individuals are under 65 years old.

Genotyping of Affected and Control Individuals

The general strategy to perform the association studies was to individually scan the DNA samples from all individuals in each of the populations described above in order to establish the allele frequencies of the above described biallelic markers in each of these populations. More particularly, the biallelic markers used in the present association study are A2, A9, A15, A22, A24, A25, A26, A30, A34, A35, A36, A38, A41, A42, A44, A51, A52, A54, A55, A56, A57, A59, A60, A64, A73, A75, A76, A85, A93, A96, A108, A111, A115.

Allelic frequencies of the above-described biallelic markers in each population were determined by performing microsequencing reactions on amplified fragments obtained by genomic PCR performed on the DNA samples from each individual. Genomic PCR and microsequencing were performed as detailed above in examples 2 and 4 using the described PCR and microsequencing primers.

Association Study between Prostate Cancer and the Biallelic Markers of the PCTA-1 Gene

The alleles of two biallelic markers, namely (T) A30 and (T) A41, have been shown to be significantly associated to familial prostate cancer, more particularly early onset familial prostate cancer. Indeed, the allele T of the biallelic marker A30 showed a p-value of 1.08×10⁻² for the early onset familial prostate cancer and of 3.39×10⁻² for the familial prostate cancer. The allele T of the biallelic marker A41 presented a p-value of 4.04×10⁻² for the early onset familial prostate cancer. These two markers could be then used in diagnostics.

Some other biallelic markers, namely A54, A55, A56, A57, A59, A60, A61, A85, A96, A108, A115, showed a moderate association. These biallelic markers are localized in the exons and introns of the PCTA-1 gene.

The inventors observed that all the PCTA-1-related biallelic markers were in linkage disequilibrium with each other in the controls individuals. In the familial cases of prostate cancer, the biallelic markers localized in the promoter did not show a linkage disequilibrium with those localized in exonic and intronic region of the PCTA-1 gene and were not in linkage disequilibrium with each other. This lack of linkage disequilibrium for the promoter biallelic markers suggests that this region comprises a trait causing mutation and could explain the cases haplotypes.

Haplotype Frequency Analysis

One way of increasing the statistical power of individual markers, is by performing haplotype association analysis.

Haplotype analysis for association of PCTA-1 markers and prostate cancer was performed by estimating the frequencies of all possible haplotypes comprising biallelic markers selected from the group consisting of A2, A9, Al5, A22, A24, A25, A26, A30, A34, A35, A36, A38, A41, A42, A44, A51, A52, A54, A55, A56, A57, A59, A60, A64, A73, A75, A76, A85, A93, A96, A108, A111, A115 in the cases and control populations described in Example 5, and comparing these frequencies by means of a chi square statistical test (one degree of freedom). Haplotype estimations were performed by applying the Expectation-Maximization (EM) algorithm (Excofflier L & Slatkin M, 1995), using the EM-HAPLO program (Hawley M E, Pakstis A J & Kidd K K, 1994).

Haplotype Frequency Analysis for Familial Cases of Prostate Cancer

The most significant haplotypes obtained with the familial cases of prostate cancer are shown in Table 5. These haplotypes comprise the biallelic markers A2, A30, A41, A55, A57, and 5-202/95.

The preferred two-markers haplotypes are described in Table 5 as H1 to H7 of PT2. The more preferred haplotype is the haplotype H1/PT2 and comprises the biallelic markers A30 (99-1572/440 allele T) and A41 (allele T). This haplotype presented a pvalue of 1.1×10⁻⁴ and an odd-ratio of 1.67. Estimated haplotype frequencies were 57.2% in the cases and 44.4% in the controls.

The preferred three-markers haplotypes are described in Table 5 as H1, H2, H3, H7, H8, H9, H10, H11, and H12 of PT3. The more preferred haplotype is the haplotype H1/PT3 and comprises the biallelic markers A2 (allele A), A30 (99-1572/440 allele T) and A41 (allele T). This haplotype presented a p-value of 1.1×10⁻⁵ and an odd-ratio of 1.84. Estimated haplotype frequencies were 42.9% in the cases and 29% in the controls.

The preferred four-markers haplotypes are described in Table 5 as H1, H2, H4, H5, H7, H9, H16, H17, H18 and H19 of PT4.

In conclusion, most preferred haplotypes for the familial cases of prostate cancer comprise the biallelic markers A30 (99-1572/440 allele T) and/or A41 (allele T). Some other preferred haplotypes for the familial cases of prostate cancer comprise the biallelic marker A2 (allele A). Optionally, preferred haplotypes for the familial cases of prostate cancer comprise the biallelic markers A55 (allele C) and/or A57 (allele G). These haplotypes can be used in diagnostic of prostate cancer susceptibility.

Haplotype Frequency Analysis for Sporadic Cases of Prostate Cancer

The most significant haplotypes obtained with the sporadic cases of prostate cancer are shown in Table 6. These haplotypes comprise the biallelic markers A2, A30, A41, A55, A57, and 5-202/95.

The preferred two-markers haplotypes are described in Table 6 as H1 to H4, H6, and H7 of PT2. The first more preferred haplotype is the haplotype H1/PT2 and comprises the biallelic markers A2 (allele T) and A55 (allele T). This haplotype presented a p-value of 2.4×10⁻⁴ and an odd-ratio of 1.94. Estimated haplotype frequencies were 16.2% in the cases and 9% in the controls. The second more preferred haplotype is the haplotype H2/PT2 and comprises the biallelic markers A2 (allele T) and A57 (allele A). This haplotype presented a p-value of 5.3×10⁻⁴ and an odd-ratio of 1.84. Estimated haplotype frequencies were 16.3% in the cases and 9.5% in the controls.

The preferred three-markers haplotypes are described in Table 6 as H1, H2, H3, H4, H6, H7, and H8 of PT3. The more preferred haplotype is the haplotype H2/PT3 and comprises the biallelic markers A2 (allele T), A55 (allele T), and A57 (allele A). This haplotype presented a p-value of 2.3×10⁻³ and an odd-ratio of 1.75. Estimated haplotype frequencies were 15% in the cases and 9.2% in the controls.

The preferred four-markers haplotypes are described in Table 6 as H1, H2, H3, H4, and H6 of PT4.

In conclusion, most preferred haplotypes for the sporadic cases of prostate cancer comprise a biallelic marker selected from the group consisting of A2 (allele T), A55 (allele T), and A57 (allele A). Optionally, preferred haplotypes for the familial cases of prostate cancer comprise the biallelic markers A30 (allele T) and/or A41 (allele T). These haplotypes can be used in diagnostic of prostate cancer.

Summary of Haplotype Frequency Analysis

The most preferred two- and three-biallelic markers haplotypes for the familial and sporadic prostate cancer are summarized in Table 7. These haplotypes can be used in diagnostic of prostate cancer susceptibility.

The statistical significance of the results obtained for the haplotype analysis was evaluated by a phenotypic permutation test reiterated 1000 or 10,000 times on a computer. For this computer simulation, data from the cases and control individuals were pooled and randomly allocated to two groups which contained the same number of individuals as the case-control populations used to produce the haplotype frequency analysis data. A haplotype analysis was then run on these artificial groups for the five haplotypes of the Table 7 which presented a strong association with prostate cancer. This experiment was reiterated 1000 times and the results are shown in Table 8.

The haplotypes 1 and 2 of the Table 7 are clearly associated with familial prostate cancer and more particularly with familial cases which were under 65 years and with>3caP familial cases. The permutation test clearly validate the statistical significance of the association between these haplotypes and familial prostate cancer since, among 1000 iterations, none of the obtained haplotypes had a p-value comparable to the one obtained for the haplotypes 1 and 2 of Table 7 for the familial cases, the familial cases under 65 years and the>3caP familial cases.

The haplotypes 3, 4, and 5 of the Table 7 are clearly associated with the sporadic prostate cancer. The permutation test clearly validate the statistical significance of the association between these haplotypes and sporadic prostate cancer since, among 1000 iterations, less than 6 of the obtained haplotypes had a p-value comparable to the one obtained for the haplotypes 3, 4 and 5 of Table 7 for the sporadic cases. Moreover, among 1000 iterations, none of the obtained haplotypes had a p-value comparable to the one obtained for the haplotypes 3, 4 and 5 of Table 7 for the informative sporadic cases.

Attributable Risk

The attributable risk has been calculated as described in the “Evaluation of risk factors” of the part entitled “Statistic method”. The results are disclosed in Table 9.

These results show that the preferred haplotypes disclosed in the present invention are highly significant for the prostate cancer. Indeed, 16.92% of the sporadic prostate cancer cases carried the haplotype 4 of the Table 7 considering a dominant model which is the more relevant model for prostate cancer. Moreover, 60.77% of the familial early onset prostate cancer cases carried the haplotype 1 of the Table 7 considering a dominant model.

TABLE 5 Haplotype frequency analysis for the familial cases of prostate cancer A2 A30 A41 A55 A57 A75 67/67 72/66 75/71 72/68 72/69 95/95 frequency % (A) (T) (T) (C) (G) (G) haplotype abs diff freq all 0,1 6,4 4,2 3,8 3,4 0 frequencies Odds Pvalue pvalue 7,5e-01 3,3e-02 1,4e-01 2,0e-01 2,5e-01 7,5e-01 cases Controls ratio (1df) Cases/controls ↓ H1 PT2 183/298 T T 0.572 0.444 1.67 1.1e-04 H2 188/298 T G 0.540 0.431 1.55 8.6e-04 H3 183/296 T C 0.536 0.428 1.54 1.1e-03 H4 183/299 A T 0.543 0.460 1.40 1.1e-02 H5 192/299 A T 0.517 0.440 1.36 1.8e-02 H6 184/300 T T 0.046 0.022 2.15 3.4e-02 H7 183/298 A C 0.518 0.451 1.31 4.3e-02 H1 PT3 181/294 A T T 0.429 0.290 1.84 1.1e-05 H2 186/295 A T G 0.406 0.274 1.82 1.8e-05 H3 181/292 A T C 0.405 0.274 1.80 3.2e-05 H7 180/294 T T C 0.506 0.396 1.56 9.1e-04 H8 179/295 T G G 0.547 0.436 1.56 9.1e-04 H9 181/293 T C G 0.542 0.432 1.55 1.0e-03 H10 181/295 T T G 0.534 0.426 1.54 1.2e-03 H11 179/291 T C G 0.540 0.433 1.54 1.4e-03 H12 178/293 T T G 0.510 0.404 1.54 1.5e-03 H1 PT4 177/288 A T C G 0.413 0.276 1.85 1.5e-05 H2 177/292 A T G G 0.415 0.278 1.84 1.5e-05 H4 179/289 A T C G 0.409 0.279 1.79 3.4e-05 H5 176/290 A T T G 0.389 0.260 1.81 3.7e-05 H7 178/290 A T T C 0.383 0.260 1.77 6.7e-05 H9 179/291 A T T G 0.395 0.280 1.67 2.7e-04 H16 177/288 T C G G 0.545 0.438 1.54 1.5e-03 H17 178/291 T T C G 0.506 0.400 1.53 1.5e-03 H18 176/289 T T C G 0.508 0.405 1.52 2.1e-03 H19 176/290 T T G G 0.510 0.408 1.51 2.2e-03 1df refers to one degree of freedom.

TABLE 6 Haplotype frequency analysis of the sporadic cases of prostate cancer haplotype frequencies Pvalue A2 A30 A41 A55 A57 A75 cases Controls Odds ratio (1 df) frequency % 60/67 64/66 73/71 64/68 65/69 94/95 (A) (T) (T) (C) (G) (G) abs diff freq. all −7,4 −2,0 2,7 −3,7 −3,9 −1 pvalue 7,7e-03 4,3e-01 2,9e-01 1,6e-01 1,4e-01 3,4e-01 Cases/controls ↓ H1 PT2 281/298 T T 0.162 0.090 1.94 2.4e-04 H2 282/301 T A 0.163 0.095 1.85 5.3e-04 H3 282/301 T T 0.140 0.083 1.79 2.1e-03 H4 283/298 T A 0.136 0.083 1.74 3.6e-03 H6 283/299 T T 0.317 0.246 1.42 7.3e-03 H7 279/300 T T 0.045 0.022 2.08 3.0e-02 H1 PT3 278/295 T T T 0.083 0.037 2.33 1.1e-03 H2 277/294 T T A 0.150 0.092 1.75 2.3e-03 H3 279/295 T T A 0.081 0.040 2.12 3.4e-03 H4 278/294 T T A 0.134 0.082 1.75 3.8e-03 H6 277/295 T T G 0.126 0.076 1.76 4.7e-03 H7 277/293 T T A 0.091 0.048 1.96 4.7e-03 H8 275/294 T T T 0.093 0.051 1.91 5.5e-03 H1 PT4 273/290 T T T A 0.046 0.010 4.76 2.0e-04 H2 271/290 T T T T 0.044 0.010 4.54 3.9e-04 H3 274/291 T T T A 0.078 0.038 2.15 3.6e-03 H4 274/292 T T T G 0.053 0.021 2.57 4.4e-03 H6 272/289 T T T A 0.090 0.048 1.95 5.5e-03

TABLE 7 Haplotype frequency analysis of the preferred haplotypes Pvalue haplo. Frequency % (cases vs controls) HAPLOTYPE familial cases sporadic cases MARKERS A2 A30 A41 A55 A57 vs controls vs controls FAMILIAL PT2 haplotype 1 T T 1e-04 (57/44) 6e-01 (45/44) CASES PT3 haplotype 2 A T T 1e-05 (43/29) 2e-01 (26/29) HAPLOTYPES SPORADICS PT2 haplotype 3 T A 4e-01 (11/10) 5e-04 (16/10) CASES haplotype 4 T T 3e-01 (11/9)  2e-04 (16/9)  HAPLOTYPES PT3 haplotype 5 T T A 3e-01 (11/9)  2e-03 (15/9) 

TABLE 8 Haplotype frequency analysis with permutation test results number haplotype PERMUTATIONS cases/ frequency Pvalue TEST SAMPLES controls cases controls Odds ratio (1 df) Iter/nb of Iter. HAPLOTYPE 1 of Table 7 cases vs controls 463/298 0.501 0.444 1.26 2.8e-02  30/1000 cases (<=65 years) vs controls 176/298 0.546 0.444 1.51 2.3e-03  3/1000 cases (>65 years) vs controls 283/298 0.467 0.444 1.10 4.0e-01 273/1000 sporadic cases vs controls 280/298 0.455 0.444 1.04 6.5e-01 572/1000 sporadic cases (<=65 years) vs controls  89/298 0.454 0.444 1.04 7.5e-01 696/1000 sporadic cases (>65 years) vs controls 187/298 0.450 0.444 1.02 7.5e-01 771/1000 sporadic informatif vs controls  67/298 0.434 0.444 0.96 7.5e-01 699/1000 familial cases vs controls 183/298 0.572 0.444 1.67 1.1e-04  0/1000 familial cases (<=65 years) vs controls  87/298 0.646 0.444 2.28 2.7e-06  0/1000 familial cases (>65 years) vs controls  96/298 0.501 0.444 1.25 1.7e-01 103/1000 familial cases (>=3 caP) vs controls  82/298 0.588 0.444 1.79 1.1e-03  0/1000 HAPLOTYPE 2 of Table 7 cases vs controls 457/294 0.325 0.290 1.18 1.4e-01 127/1000 cases (<=65 years) vs controls 174/294 0.362 0.290 1.39 2.1e-02  24/1000 cases (>65 years) vs controls 279/294 0.297 0.290 1.03 7.5e-01 770/1000 sporadic cases vs controls 276/294 0.257 0.290 0.85 2.1e-01 176/1000 sporadic cases (<=65 years) vs controls  88/294 0.229 0.290 0.73 1.1e-01  99/1000 sporadic cases (>65 years) vs controls 184/294 0.265 0.290 0.88 4.0e-01 351/1000 sporadic informatif vs controls  67/294 0.176 0.290 0.52 6.9e-03  9/1000 familial cases vs controls 181/294 0.429 0.290 1.84 1.1e-05  0/1000 familial cases (<=65 years) vs controls  86/294 0.501 0.290 2.46 2.5e-07  0/1000 familial cases (>65 years) vs controls  95/294 0.365 0.290 1.41 4.8e-02  48/1000 familial cases (>=3 caP) vs controls  82/294 0.467 0.290 2.14 2.0e-05  0/1000 HAPLOTYPE 3 of Table 7 cases vs controls 470/301 0.143 0.095 1.58 5.8e-03  15/1000 cases (<=65 years) vs controls 175/301 0.132 0.095 1.44 7.8e-02  96/1000 cases (>65 years) vs controls 292/301 0.145 0.095 1.61 8.2e-03  14/1000 sporadic cases vs controls 282/301 0.163 0.095 1.85 5.3e-04  4/1000 sporadic cases (<=65 years) vs controls  90/301 0.163 0.095 1.85 1.1e-02  11/1000 sporadic cases (>65 years) vs controls 189/301 0.158 0.095 1.77 3.4e-03  10/1000 sporadic informatif vs controls  70/301 0.221 0.095 2.69 3.4e-05  0/1000 familial cases vs controls 188/301 0.110 0.095 1.17 4.4e-01 487/1000 familial cases (<=65 years) vs controls  85/301 0.096 0.095 1.00 7.5e-01 991/1000 familial cases (>65 years) vs controls 103/301 0.121 0.095 1.31 2.7e-01 317/1000 familial cases (>=3 caP) vs controls  83/301 0.074 0.095 0.76 3.7e-01 462/1000 HAPLOTYPE 4 of Table 7 cases vs controls 464/298 0.143 0.090 1.68 2.2e-03  7/1000 cases (<=65 years) vs controls 174/298 0.135 0.090 1.57 3.0e-02  47/1000 cases (>65 years) vs controls 286/298 0.145 0.090 1.70 3.8e-03  9/1000 sporadic cases vs controls 281/298 0.162 0.090 1.94 2.4e-04  2/1000 sporadic cases (<=65 years) vs controls  88/298 0.165 0.090 2.00 4.7e-03  17/1000 sporadic cases (>65 years) vs controls 189/298 0.156 0.090 1.87 1.7e-03  4/1000 sporadic informatif vs controls  69/298 0.223 0.090 2.89 1.1e-05  0/1000 familial cases vs controls 183/298 0.110 0.090 1.25 2.9e-01 318/1000 familial cases (<=65 years) vs controls  86/298 0.100 0.090 1.12 6.5e-01 726/1000 familial cases (>65 years) vs controls  97/298 0.120 0.090 1.37 2.2e-01 271/1000 familial cases (>=3 caP) vs controls  81/298 0.084 0.090 0.93 7.5e-01 839/1000 HAPLOTYPE 5 of Table 7 cases vs controls 456/294 0.136 0.092 1.56 9.1e-03  14/1000 cases (<=65 years) vs controls 171/294 0.131 0.092 1.48 6.5e-02  80/1000 cases (>65 years) vs controls 282/294 0.136 0.092 1.55 1.8e-02  30/1000 sporadic cases vs controls 277/294 0.150 0.092 1.75 2.3e-03  6/1000 sporadic cases (<=65 years) vs controls  88/294 0.155 0.092 1.81 1.7e-02  27/1000 sporadic cases (>65 years) vs controls 186/294 0.142 0.092 1.64 1.5e-02  34/1000 sporadic informatif vs controls  69/294 0.226 0.092 2.89 1.0e-05  0/1000 familial cases vs controls 179/294 0.112 0.092 1.24 3.2e-01 354/1000 familial cases (<=65 years) vs controls  83/294 0.102 0.092 1.12 6.5e-01 733/1000 familial cases (>65 years) vs controls  96/294 0.121 0.092 1.36 2.4e-01 262/1000 familial cases (>=3 caP) vs controls  79/294 0.082 0.092 0.88 6.5e-01 749/1000

Familial forms in which at least three persons are affected by prostate cancer in the family are described in the present application as>3CaP. Sporadic cases were classified as informative sporadic cases when at least two sibs of the case both aged over 50 years old are unaffected.

TABLE 9 Attributable risk for prostate cancer Estimating of haplotype Dominant Model Recessif Model frequency Carriers Carriers Random frequency frequency Sample sizes Controls controls (cases vs Attributable (cases vs Attributable cases vs controls cases (unaffected) (French) controls) Risk % controls) Risk % Haplotype 4 281 vs 298 16.2%  9% 10.3% 30% vs 17% 16,92 3% vs 1%  2,38 of Table 7 Haplotype 1  87 vs 298 64.6% 44%   48% 88% vs 69% 60,77 42% vs 20% 30,63 of Table 7 CARRIER: Individual carrying the haplotype ODD RATIO of Carrier (OR): Carrier of cases * (1-Carrier of controls)/Carrier of controls * (1-Carrier of cases) ATTRIBUTABLE RISK (RR): Carrier of Randoms controls * (OR − 1)/(Carrier of Randoms controls * (OR − 1) + 1) (ref: Epidémiologie - Principes et méthodes quantitatives - J. Bouyer, D. Hémon, S. Cordier 1995)

Example 6 Mouse PCTA-1 Protein

The inventors have cloned a cDNA molecule encoding a mouse homologue of the PCTA-1 protein (SEQ ID No 8). The deduce amino acid sequence is provided in SEQ ID No 9. FIGS. 7A-D show alignments between the human and mouse PCTA-1 protein sequences of the inventions, as well as that of GenBank L78132. It shows an 80% homology between the human and mouse homologues.

Further comparisons between these mouse and human cDNA and protein sequences, taking into consideration the position of significant polymorphisms in relation with potentially conserved motifs, should allow the person skilled in the art to identify regions of specific physiological interest, in the design of suitable vaccine or therapeutic candidates. Two galactoside binding sites shown in FIGS. 7A-D are of special interest. These sites are conserved among the PCTA-1 proteins and the galectins, and seem to be involved in the cell-cell and cell-matrix interactions which are of high relevance to cancer. Two other sites, HFNPRF and VVCN, are also highly conserved among all these proteins.

Example 7 Preparation of Antibody Compositions to a PCTA-1 Protein

Substantially pure protein or polypeptide is isolated from transfected or transformed cells containing an expression vector encoding a PCTA-1 protein or a portion thereof. The concentration of protein in the final preparation is adjusted, for example, by concentration on an Amicon filter device, to the level of a few micrograms/ml. Monoclonal or polyclonal antibody to the protein can then be prepared as follows:

A. Monoclonal Antibody Production by Hybridoma Fusion

Monoclonal antibody to epitopes in a PCTA-1 protein or a portion thereof can be prepared from murine hybridomas according to the classical method of Kohler et al. (1975) or derivative methods thereof. Also see Harlow et al. 1988.

Briefly, a mouse is repetitively inoculated with a few micrograms of a PCTA-1 protein or a portion thereof over a period of a few weeks. The mouse is then sacrificed, and the antibody producing cells of the spleen isolated. The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the system on selective media comprising aminopterin (HAT media). The successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued. Antibody-producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as ELISA, as originally described by Engvall, (1980), and derivative methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Davis et al.

B. Polyclonal Antibody Production by Immunization

Polyclonal antiserum containing antibodies to heterogeneous epitopes in a PCTA-protein or a portion thereof can be prepared by immunizing suitable non-human animal with this PCTA-1 protein or a portion thereof, which can be unmodified or modified to enhance immunogenicity. A suitable non-human animal is preferably a non-human mammal is selected, usually a mouse, rat, rabbit, goat, or horse. Alternatively, a crude preparation which has been enriched for the PCTA-1 concentration can be used to generate antibodies. Such proteins, fragments or preparations are introduced into the non-human mammal in the presence of an appropriate adjuvant (e.g. aluminum hydroxide, RIBI, etc.) which is known in the art. In addition the protein, fragment or preparation can be pretreated with an agent which will increase antigenicity, such agents are known in the art and include, for example, methylated bovine serum albumin (mBSA), bovine serum albumin (BSA), Hepatitis B surface antigen, and keyhole limpet hemocyanin (KLH). Serum from the immunized animal is collected, treated and tested according to known procedures. If the serum contains polyclonal antibodies to undesired epitopes, the polyclonal antibodies can be purified by immunoaffinity chromatography.

Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species. Also, host animals vary in response to site of inoculations and b dose, with both inadequate or excessive doses of antigen resulting in low titer antisera. Small doses (ng level) of antigen administered at multiple intradermal sites appears to be most reliable. Techniques for producing and processing polyclonal antisera are known in the art, see for example, Mayer and Walker (1987). An effective immunization protocol for rabbits can be found in Vaitukaitis, et al. (1971).

Booster injections can be given at regular intervals, and antiserum harvested when antibody titer thereof, as determined semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations of the antigen, begins to fall. See, for example, Ouchterlony, O. et al., (1973). Plateau concentration of antibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12 μM). Affinity of the antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher, (1980).

Antibody preparations prepared according to either the monoclonal or the polyclonal protocol are useful in quantitative immunoassays which determine concentrations of antigen-bearing substances in biological samples; they arc also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample. The antibodies may also be used in therapeutic compositions for killing cells expressing the protein or reducing the levels of the protein in the body.

While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein by the one skilled in the art without departing from the spirit and scope of the invention. Accordingly, the scope of this invention is intended to be defined only by reference to the appended claims.

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22 1 106746 DNA Homo sapiens misc_feature 1..68647 5′regulation region 1 ttggcttggc agggcaacca gctcaccaga ctctctgcag acccgaagtc attacataca 60 gtatgataac agggaatgga cccgaccagc atttgctgga gatgatatct ggtgtcagcc 120 cgacaggccc ctacctgctt ctcttgatat gcaggaatcc cttcaagctc caacaagatc 180 tgtttaatag actggagagt cctttagttc cttcctctaa gggaaaatca gatcgttctg 240 gtttgcttgg taactcctta cttcatccct gatgggaagt ttatagaatg aggaaccagg 300 gctattacat gaaactataa aactgcctag agcacatact tggtattttt aacattgttg 360 agagggactc acttaattca gccttgcagc tattgcattc ctgtccaaac caacggcagg 420 ttctcaaaac aagcggtgaa agggttcctg ttgcagagct gtctggacat ttaaagaagg 480 gagaggaaat ctcaaggggt cggttgcact ggaatagaaa tcgcctgttc tttttttttg 540 agacggagtc tcgctctgtc acccaggctg gagagcagtt gcgcgatctt tgctcactgc 600 aacctctgcc tcccgggttc acgccattct cctgcctcag cctcctgaat agctgggact 660 acaggcgccc gccaccacgt ctggctcatt ttttgtattt ttagtagaga tggagtttca 720 ccattttagc caggatggtc tcgatctgct gaccttgtaa tccacccgcc tcggcctccc 780 aaagtacagg gattataggc gtgagccacc gcgcccaggt gcctgttcct tttttaagag 840 tctcactctg tcgcccaggc tggcgtgcag tggcgcgatc tctgcttact gcagtctccg 900 tctcctgagt tcaaatcaag cgagaaatca cttgttctct tctgtgaacg gaagcatcgc 960 agatctctct tggcctcaca ctcctccatc tccctgattc ctctgttctt catttaccta 1020 ccttcccagc agtctgcaga gctggccgct cactcacctc tagtaagggg atggagggtc 1080 ctgtgttgga ataactcact gaccgctaga aagttaaaaa taaatgggta atgccaggag 1140 aacttggctg gtgccttaaa agccatagaa cttctctttc catctgtaga taactgtaga 1200 caattttgtc caaaacagat aatgatctga ttctacctcc cattggtatt tcccttcctc 1260 ggcctgtgac atctcacttt ctctagactg aactttatcc cagactgtga ccttgccatg 1320 accttcctcc tgcgtgtgcc tctgccacca caggaatggc cacgcctcag atcatgtcac 1380 cgctgggaac aaaccctcta cctgcgactc tgaagttccc tctctgaccc tcttttcttt 1440 cttcccctcc cccctcccct cactccctct gcacctgtgt ttcgctgtca cgctcccaac 1500 tcatccctgt agagctggtg aagagatgct gatgtagttc ttgaccttga accccagccc 1560 tgcagccgtc ctgtggcctc actgacccag cgtcatgccc tggtcaagca ttttggtgat 1620 gctcttggtg attttcaatg ggacctgcct tgccaagccc tgggcttagg tgaaccagga 1680 ccacctgcat tctatgtttt tgattgctgg aaaaaaatca tgaaatgtca actgttgttc 1740 tcatttttcc cactgccagt tcctgctacc caacctccgc cctcatttca aggccttgag 1800 tacttttttt ctatagtgaa gtctcccaaa aatgatattt ttttaaaaaa gaaaagccat 1860 agtactctga tttgatgtgg tctgttaata cctatgggct ttgacttgtt tctgctttta 1920 gacctagaca aaataaaata tctgtggtaa aacatattca agtttaccgg gcacgggggc 1980 tcacgcctgt aatcccagca ctttgggagg ctggggcagg cagatcactt gagcccagga 2040 gtttgagacc agcctgggca acagggtgaa acaacatctc tacaaaaata caaaaaatac 2100 ctgggcattg tggtgcatgc ctgtagtcgc agctactcgg gagactgagg tgggaggatg 2160 gcttgagctc tggaggcgga ggtcatagtg agccaagatc gtgccactga actccagcct 2220 gggcaacaga ggcagattct ttctctctaa aaaacataaa ataaaaaaag gccaggcgca 2280 gtggctcaca cctgtaatcc cagcactttg ggaggctgag gggggcggac gaagaggtca 2340 ggagatagag accatcctgg ccaacatggt gaaaccctgc ctctactaaa aatacaaaaa 2400 ttagccgggt gtagtggtgc atgcctgtaa tctcaactac tcaggaggct gaggcaggaa 2460 aatcgcttga acccaggagg cggaggctgc agtgagccaa gatcgcacca ctgcactcca 2520 gcctgggtga cagagcaaga ctctgtcccc caccaaaaaa aataaataaa taaatcaggc 2580 caaagggcaa aaatgcttgc tttttagcac ttagtagtta tttccccaag aagagcggga 2640 gagaagttta ttaataatga aactggacag ttctttatca gctctaattg tttgactcaa 2700 tggcttctct tctcattacc atgcagtgct ctgctggctg caatgccttt gaacttcaca 2760 agaaggttag aatttcactg agacattcgg atggtgtggg tgtcagggtg cagctctcac 2820 acatagttga gagtgtaaat tgatacaact ttatggaaaa ttaattggga gtacccattc 2880 acactcctgt ctagcaatct cactttaagg acttgatcct acagaactca ttacatggtg 2940 caaggttcac agtgtggcat tcaaaataga gaagagctgc gggtaactcc catgcccgtt 3000 ggcaggaact ggttgaataa attatggtgc atcagtgctg tggggtatca ttaaaccatt 3060 aaaaagaaga gagagtcctg gccttaaaaa aaacttatct gatgtattgt taaacagata 3120 aagcaagttg tagatcaatg tgatttgggg ctaaaaaaat atttctatat aggtgtgaac 3180 atggccatga ctaaggaatc aggaaggaag tacctagatt gtaaccagta acatgtcggg 3240 agtgagatgg gattgagaga cgtaataata gattgagaga aaaagatttt cccatctctt 3300 tttgattttt taagaaaaca gcatgatttt cagtaatttt tacttttgtg tgtttttggt 3360 attttttctt tttctttttt tttttttttt ttttgagacg gagtttcact cttgttgccc 3420 aggctggaat gcgatggccc agtcccagct cactgcaacc ttcacttccc aggttcaaga 3480 gattctcctg tctcagcctc ccgagtagct gggattacag gcccctgcca ttacgcccag 3540 ctactttttg tatttttagt agagatgggt ttcaccgtgt tggttaggct ggtttgaact 3600 cctgagctca ggcgatctgc ctacttcagc ctcccaaaat gctgggatta cagccgtgag 3660 ccaccgcccc cagccggtat tttttcaaat caaagaaaaa ataatagagt aaatcatcca 3720 aaactttaga tggtatttag actcagtaaa cttttcatat atgacagatg aagccaaatg 3780 gtctttctgt gcagtcagct agcacacaat tgtgcacccg aggaaaatta gagactgaac 3840 cggggtgtct gtggatgcat ttcctcagca ttcagccttc cttttgcccg tgttctagca 3900 ttacttctgt cctacagcct gggatttgtg aatgaaatag acaggtgcaa aaactccctg 3960 cctgtctgta atatccatag ccccgtgctc tacttgtatt tgcatgtaca aaccataatc 4020 tcctgtaaaa tactctgtga tatttctgaa taataataaa ctctacatcc tacacaaagg 4080 caaaacccct gtatctttca tctttgaaac catagcaaag gtatgaaatt acacctgagc 4140 atgcctggcc tcaaagtcct ggaacggtta tgtctttgac cctcacttca actcaactcc 4200 agaagaagca ggtcttcctt gtaattggat agaaaactca ttgtagagaa gaaagatcta 4260 caggtcaaga aacccacagg tttgctgtaa tccgagcaaa gcactgtagc atttatttta 4320 tattttcact cttcttattt agctcttttt tttttttttt ttttgagatg gagtttcatt 4380 cttgtcaccc agcctggagc aatggtgcca tctcggctca ctgcaacctc tgcctcccag 4440 gttcaagtga ttctcctgcc tcagcctcct gagtagctgg ggttacaggc tcccaccgcc 4500 acacccagcc aattttttgt atttttagta gagacggggt ttcaccatgt tagccagact 4560 ggtctcaaac tcctggcctt aggtgatcca cccgcctcag catcccaaag tgctgggatt 4620 acaggcgcac cggccttagc tcttttatcc ttaatgaaat gctcctcatt ccctgaggtc 4680 tcacttgaat tcttgcccac ctctgggttg ccttcctctt ctgtctgtgc tttgtaacac 4740 gtggttcctt atgatgtcaa tatttatgca tatgtcttca ttccattact ggattataat 4800 cttgaagcaa cagatttttg tctctatatc ccagagccta gaatggattc ttacactggg 4860 cagtaagtac ttaataaatg tatcccaaat caaataaata catttcttct ttttcttttc 4920 tttttttttt ttttttgaga cagggttcca ctctgtcacc caggctggag tgtaatgaca 4980 tgatctcagc ttactacagc ctcaatctcc tgggcttaag caatcctccc acctcagcct 5040 cccacatagc taggactaca ggcgctcacc acaacacctc atttttgtat attttttgta 5100 gagattgggg gatctcacta cgttgccccg gctggttttg aacttctggg ctcagacaat 5160 ccacccacct tggcctccca aactgttgag attacaggaa tgagccacca ttccctggcc 5220 aaatacattt ctaaaagcca gtttctggag tatactgtca aataatagat atatgtccac 5280 atttttatac ggacttatat tgtaagaaaa agtaaaaata agtgtgaagt tattacagta 5340 atagtaatta ttttgcagaa aaagaactga gtttaaacag gctttttaga aaaacccaac 5400 aggagattca cagtctggta ctaacgttta gacatggatc atcagtaaat gtgttccaaa 5460 gagttacaca gataccagct ttgtcttggg aattcttacc cctgaaaatt gattgactat 5520 cactgactgt gtgacatgag aaagttttgt ggggtttttt ttttgtattt ttttgagacg 5580 tatcttgctc tgtcacccaa gctggagtcc actggcgcga tcttggctca ctacaacctc 5640 tgccgcctgg ttcaagcgat tctcctgcct cagcctccag aatagctgcg attacaggca 5700 cctgccacca tgcccggcta atttttgtat ttttagtaaa gacggggttt catcgtgttg 5760 gccaggatgg tcttgaactc ctgacctcag gtgatctgcc cacctcagcc tcccaaagtg 5820 ctgggattac aggcatgagc caccgtgccc agcctgaaaa agttttgaac ggtctaaatc 5880 catatgctgt gaatcctatt accatcacac acttaggcat ttaaaatcat attttcaagg 5940 ccaggtactg aaatattttc tgcaagcaga gagatcaaac tttagcattg ttattcttgt 6000 agtagtttca tagtttgagg tcttagattt aagtcttcca ttgattttga tttgattttt 6060 gtatatggag ataggggtct agtttcattc ttttgcatat ggatatccag ttttcccagc 6120 accatttatt gaagagactg tctttttcac cagtgtatgc tcttggcacc ttcgtcaaaa 6180 atgagttccc tgtaggtgtg tgggtttgct tctggttctc tattctgtcc cattggtcta 6240 agtgtttggt tttatgctac taccatgctg gttggtatag ctctgcagta taacttgaaa 6300 gcaggtaatg tgattccaaa gaagctagtt aagtaattga gctaaactgg aacctcaggt 6360 gtagaagtca taagcgtggg gagcgtttct tctcaggttc tctgcctata atttagtttg 6420 ccacaccaga tgaacagtga caacttggtc ttggtgttcg tggtggtttc caaccaaact 6480 ttggtcataa caggtgaacc agcctggggc atgctttccc attcggttat cctccccata 6540 gtttgcaaag tagcaaagat gaactcttca tgagttggct aagcatagac atttcaagac 6600 caaactaaac gtcctgaaga gcatgtttca cagaaaacta gcccctaagg gaccagtggg 6660 ggctgtcaga gaacaaggtt tcaacgtact gagttttaaa gatctaattg gcttttaata 6720 acaattcatg aaccaggcac catagtctac aaaatagaca gggtttctgc tgggcactgc 6780 aggacagttg gtttttggaa ggtggcttga gcaggaacaa ggaaaaagca ccgtgccaag 6840 agtggattgg ttaacatcag gggacttcgg gtgactttcc ttctatgggt taaagcaaag 6900 gggacttccc tagcatgtca gctcaggttg actgggcccc tttggattgg ttgctgtgaa 6960 tctcctagtt ttttttgttt tggtttggtt tgggtctttt ggggaaaacg ggccagtttg 7020 gagattcagc tattatttct ctctcctgat atcagaagat cagatcttat gagtacacag 7080 ctgaggtttt gggttggtga tgtggaaccc tggtgtgagt gactccattt tgggttggtc 7140 tattggggtc tcggtgcagg agctcagtcc aaatcagtgg cctctcctca tttttatttg 7200 acttctccat caatctatcc gtgtctcccg tcacatcagt ccattccccc gtgggctgca 7260 cattcagctc ggagctgaga gcttttccca gggtgtgccc tggggtttct gctgcttgca 7320 gcctgatatt aaatctcagg tgtaaatctt cagaggcaac tgttccttag tacccagagc 7380 tttcagctcc ctgagcagaa atgggacttg actgtcagtt tataaactaa ccaaggtgtg 7440 aaattcatgc aacttagccg actttctgtt caaagaattc ttggcagcag ttaatacatt 7500 ttgcccaaat ataagataat tcccttgtac tcacaatgag aaagttttac aaaatggggg 7560 ttttctttag tttacttgaa tataaaacat aggtgttcca ctctgcagta ccttaacagt 7620 tcttaaggag atgtttgaaa caacccatgt ccaggcctca cacctcgcca attaaataaa 7680 tgagaagttc ttcccagcca gtgttaagaa aaattaacat caagttttag gaaggtagac 7740 agattatgca aatgcatacc tatatgattt aagttattac attaatttac acacacatat 7800 ttaaaatcat agattaatct aatttagaga tgctgcattt tttccatctc tcctgtttca 7860 taaatgttat tcacacggca tttctctgct atcctcggaa tagtgtttgt atcgtgtcac 7920 tctggcacgg ggctctacag aacatgtcga gcgtgttgcc ttccctactg cccacatcgt 7980 ttgagagaac acattttaaa cattttttta ttgtggtaaa atacacataa cataaaagtt 8040 acgattttaa ccttttttaa ctctgtcatc caggctggag tgcagtggcg agatcttggt 8100 tcactgcaac ctccgcctcc taggtccaag tgattctcct gcctcagcct tccgagtagc 8160 tgggattaca ggtgcacacc accacgnccg gctaattttg tatttttagt agatgcgggg 8220 tttcaccatg ttagccaggt tggtctcgaa ctcccgacct caggtgatca gcccgcctcg 8280 cctccccagt gctgggatta caggcgtgcg ccactgtgcc gggcccattt taaccacttt 8340 taagtgcaca gttcagtggc attaagtata ttcgcggtgt tgtgcgaccg tcaccaccat 8400 tcacctccag aacttctctg tcttcccaaa ctgaaattct gtacccattg aacggtaact 8460 ccccattccc catttctgct tcctaggccc tgacatggag gctgggccaa cggatatctc 8520 acctcccttc aggcttctcc agatttgccc ccgtttttct ccctctttgt cccatctcca 8580 aagaaatggt gtcttttcat catcaaggtc catcccttgc tccttgaata cactccaggc 8640 ccagtggaac aggcatcctg tggggtgcac ggacagggtg cctggggaac acccagggca 8700 cagaacccag accgggggtt tggagaaggt gtcctagcag aagtgatgtc taagctgagg 8760 ccctacagat aagagaaagt aagcagatga aagggctggg gagggtggca tttcaggcct 8820 acacaaccac acgcgtgttc ttcagccatc tccatggcct cactgcccac ctggtatcag 8880 ccggccacca cccggctaga acggctttca aaatcgctgc tcgtctactc ctcaccaaat 8940 cttgtcttca cttggtgctc aagcccatca cctttctgca agtattattt tttttttttt 9000 ggagatggag tctcgctctg tcacccgggc tggagtgcag tggttcaatg atagctcact 9060 gcaaccttga actcctgggc tcaagatcct cttgccacag cctcccaaag tgctgagatt 9120 acaggcacaa gccaccatgc gtggtccttg ctgcaacttt tttttttttt tttttttttt 9180 ttttgagaca gaatctcgct ctgtcgctca ggctggagtg cagtggtgtg atctcggctc 9240 aatgcaacct ccgcctcccg ggttcaggtg attctcctgc ctcaccctcc tgagtagcta 9300 ggaacacagg cgctcaccac cacatccagc taatttttgt gtttttagta gagccggggt 9360 tttgccatgt tggccaggct tctctcaaac tcctggacct cgggcgattg gcccgcctcg 9420 gcctcccaaa atgctggaat tacaggcatg agccaccgtg cctggccatt tgctgcaact 9480 tttgacactg ctccccctgc ttttcttccc ctctctgacc tcctttctct gctgtccttt 9540 cgttccttcc tctgccactg aagtgtcctt ctcaggtcct tctcaaggtt gtgaccttac 9600 agctgtctct tcacttccag tcatttcttt cataatcact ttgacatcct tattttcatc 9660 tcctgccctg gcctctccca gggaccagga ccatgcattc agctcctggg ggcatctcaa 9720 gcttgttgtg tgtgagcctg cccttgttgt cttctccgtc acctcttcac agcttgctct 9780 gcatttcacc tcctttcctg ttttccccag tgatcgcatc tctacagcgg ctctcacttc 9840 atccccttct ctcctagagg agtgatgcgg agtctcatta atccttgctt atgtcattct 9900 tcccccttct ctgtccatca cctccacatg tcctgttccc ccatgcgtcc tacactgtag 9960 ccaggtgggt atttcctgtg ctggtcttag acaccccctg aggataccct gcttcaggcg 10020 agagccctca gtgactccct gttgtccgga atgacgtcca gctccttgga cagtccccag 10080 tgtattcacc tgtctcatct ccttcttttc gttttgtttg tttttcttaa cttccagccc 10140 gatttctgaa tcatctccct cttgcccctc ccattgcctt tgcttaagac taaatgctcc 10200 ttcctcccaa gtccccactg cccagatttc agcagggtcc atctcaaaca tgtctgtctc 10260 caagaaactg cctctgattt ttttcataag aagacacctg tcctctctga cttcatctgt 10320 acccctctct tggaagtcac tatcttgtgc cttgcatttt cgttgtttaa gtggtctcca 10380 tttcccagca tatcttgagg tcaagggttc aggtcatttt atctttgtct atgcattgca 10440 atatgggggt ttttacatat tagctgctca ataaatcggt gttgaataaa ggcatgtgta 10500 tgctttcatt aagactatga aacccacaaa aatcagtggt tttcctattt cacccttaga 10560 aaacaaaccc acaacatagc acaacctgat attcagagct aagaacaaag gtcatgcata 10620 ttaatctaaa ttctatcttt atcaactttc acaagtaatt cgtatttccc tgtctgcatc 10680 acggggatga ttctggccag acattgacct tggtaaaatt tcctccagat tatgagaaat 10740 caagtcaaat atgccaagta acatagtttc tacttagagt caggttcatg ttttagcagg 10800 aacctcaaat accacaaaat ctgtcaagtt ctaacatttg tatctctcga cagtacctga 10860 agttcctgtt tctgtttcct cagcccaggt ttccaattca gtgagcagaa cggtgactgt 10920 gttggtaaaa gagcccacat acctgcccga tcctgcagga gtgttgcaga tgcaaacagg 10980 cgggtctcca catgacctgc ggagtaatga ctagtgtccc taaagtcatg gggcttctgg 11040 ggttagcctt gaaaaaagct aaaggttgca tagagagaga tttctatccg ttcagagact 11100 cactataatt ctctctttct gtctctgtcc ttcatctgtt tctctctttc tctctcactc 11160 tctctctctg atacacacac acacacacac acacacacac actcacactc acacactcct 11220 gagtaaggga aatgtgagaa gaaggtaaaa cttcaactaa atgaaaagaa attgtatgaa 11280 ttatggtaag caggttggtt tttagttcca gtaaagatag aaatatttag attacttagg 11340 agaaaagtct agctggtaac acatgggaat gtgcctgtgt gaaaacaaaa caaaacaaaa 11400 aatctaggct tgtggttagg tgaaggtatg tacactgctg agacatggcg atgggtgagc 11460 ttgggatgag gagaaaggct tctctgagaa gattaagaga gaaagattgt ttaaaaatgt 11520 ttaaacatgc tgggcactgt ggctcacacc tgtaatccca acactttggg aggccaaggt 11580 gggcggatca tgaggtcagg agttcgagac catcccggcc aacatggtga aaccctgtct 11640 ctgctaaaaa tacaaaaatt agccaggcgt ggtggcgggt ggctgtagtc ccagctactt 11700 gggaggctga ggcaggagaa tggcgtgaac ccaggaggcg gagatgcagt gagccgagat 11760 tgtgccactg cactccagcc tgggcgacag agcaagactc cgtctcaaaa aaaacaaaaa 11820 aacaaaaaaa aaacacacat tgacaccagg acggagttag cacatcttta caggtgagac 11880 tctcagaccc gagaaaatag aggcacttta gagctgagct aatcccacag ccacctcaac 11940 acacaaacgg ggaatctgag acccgcattg gcaccgtgcc tgaggttcta aagcccaggg 12000 cttctgactc gcctcttgtg cttcttcagt actgtgggtg ggggtggggt ggggggtgac 12060 attagctgat gagaaagatt ttggttttag aaagatggag ttaacataaa cgaaggtgta 12120 ctgggactgg tctcctctgc tgacttcatg ggaagcacac acacgcacac acacacacac 12180 acacacacac acacacacac atacacacac ctgtccaaga tcagaaaaaa tccctcacat 12240 ccctgtagca tgatcctgat tgtaaaaatg gagccctaat cagaagggca gaagcatgat 12300 tgcctctcaa gagatttgga cgccactttt tcatagttgg ttttagctgc tttgcgatat 12360 atactgaaat aaatagaaaa gggaaagaat tgtaacctgg attgacagac aacaagccct 12420 gacagacaaa aagcagataa gaaataaaat aaggaagata acccataatg taaaataaaa 12480 atagcacatt gttgcatgca ttgataccct tttttttttt tctttgagat cttgctctgt 12540 ctttcaggcc gaagtacagt gtctcaatca tagctcactg cagcctccag cttctgggct 12600 caagcaatct tcccatctca gccacccaag tagctggggc tgcaggcacg aactatggtg 12660 cccagctgat aatttttaaa aatagggaca ttagtgcatt tagcaaattt gagtgtctgc 12720 tgtgtatcaa gcactgttct gggcactggg acagcacagg gagcaaataa acaaaagccc 12780 ctgcgctcaa ggtgctcgta ttctagaggg agatgctgag ttcacctccc attaaaatgc 12840 cattctcaag atccagtccc tccacccacc ccagccccca gggttttggt ggaaatttaa 12900 ctaagttgga agattgataa tatctccatt cacatttgga tatgatttta atgaaggttg 12960 ctttttggtt tttagggaga agaaaatggc tttccagata gcactggaga tcctcttcca 13020 ggtaaatgat tgattctaaa gctatctggg ctaatagcta gtgtggctga ataaaagata 13080 atttgaggcc agggtcggtg actcatgcct gtaattccag cactttggga ggccaaggtg 13140 ggcggatcac ctgaggtcag gagttcaaga ccagcctggc caacatggta aaaccccgtc 13200 tctaccaaaa atacaaaaat tagctggttg tggtgggcgc ctgtaatccc agctactcgg 13260 aggctgaggc aggagaatcg cttgaacccg ggaggcggag gttgcagtga gccaagatca 13320 caccactgca ctccagcctg gacaacagag cgaaactcca tctcaaaaaa ttaaattaaa 13380 taaaataaat aatttgagac tatgtttatc attaacttta aaatctgtac tgcagaatag 13440 agcaactttc tacctgcggt gcactgcagg gaaagccgta tcttacaaga cttcacaaaa 13500 gccttcaaag agtattttct ctgcactaac cttcctttgc atgtgagggg cacggcaggg 13560 ttctgaatgg ggcaggttta ggatcaggcc agtcgggact gagtggattc ttcttccctc 13620 tgagttctaa gagccatagc attggtggag aacatgctgt ttgttgcttg gtggaaggga 13680 ccagaagcca gctgggtcat ctctctgttt gtgccttggc cacttaggta gccaaaggag 13740 ccctcctgac attaggtcag gtgttagtcc ctctcctttt ctgcttttag tgtgtttaag 13800 caaataaaca ttaaagttca tttctccccg ctcccctttt ttaatcataa gacagacatg 13860 tttgcaatgt ttaaatttct cattaatcag aagggatagg gagtgaggga gtaagcatta 13920 aaataagcta gcaaatggcc aggtgtggtg gctcacacct gtaatcccag gactttggga 13980 ggccaaggtg ggcagatcac ttgaggccag gagttcaaga ccagcatggc caacatggca 14040 aaactccatc tctactaaaa atacaaaaat tagccaggcg tggtgatggg cacctataat 14100 ctgagctact cgggaggctg aggcagagaa ttgcttgaac ccgggaggca aagattgcag 14160 tgagctgaga ctgcaccact gcattccagc ctgggtgaca gagcaagact ccatctcaaa 14220 aaaatgctag caaaataata ataataataa taataaaaca tacctcacca acattttcta 14280 catcttgtaa agcatacatt gactgactga agtcaccaga gttttgtttc tttctttctt 14340 aagcagggtg gggaacccgt agagccctca ggggcagcta tcatcagccc aggtaaccaa 14400 gctgaaaaac cagaaggtgc agtgcgtact caactttttc cccttagaaa cacgatatta 14460 gaaaatacac caataccaac atgtgagcaa cagttctctc tggaaggtgc agttctgggt 14520 gatttttttt tcattccata gatttttttt ttcttgagac ggagtttcgc actcttgttg 14580 cctaggctgg agtgcaatgg tgcgccacca cgcccggcta atttttgtat ttttagtaga 14640 gacggggttt caccatgttg gccaggctgg tctcgaactc ctgacctcag gtgatccacc 14700 tgcttcggcc tcctaaagtg ctgggatgac aggtgtctca ctatgttgcc taagcttttc 14760 tcgaacccct gagctcaagc ctcctcccac ctcagccatc caaagtgctg ggattacagg 14820 catgagccac cacgcctggt gagtttttat tttctttcca ctatccntat atttctaaaa 14880 tttctaacat gagctggtat cagaactgcc cctccgcatt taatctgtgt atacaaatgt 14940 atatataaca aatgatcaca tgttggtaan gtataccttg ctgcatggtg aaataaccaa 15000 ggaaacttct aaaaggttaa ctgtggttgg cctgggtaat gggagcatta attttttcca 15060 tatgctcatc tgaattttca gatttgctat gacaagcaca tatttatttt ctaattttaa 15120 aaatctatat ttaaactctt taaagactaa caccctacac actaatgtgg cacgttagct 15180 aaaataaaaa taaatacaga aatttgttta gaaatatttg taaacccttc aaggactctt 15240 ctgaatgata gtcattatta attagcaggt taattttaat caggcttctg gtcatcttca 15300 aacatttttt acttgtgtca aaatgaacca ccagagtgtg ggtttttttg ttattttttt 15360 tgtttttttg agacagagtt tcactcttgt tgcccaggct ggagtgcaat ggcgagatct 15420 cggctcactg caacctctgc ctcctgggtt caagcagctc tcctgcctca gcctcctcct 15480 gagtagctgg gattacaggc gcccaccacc acacccagct aatttttgta tttttagtag 15540 agatgggttt tgccatgttg gccaggttgg tcttgaactc ctcacctcag acgatccacc 15600 cacctcagcc tcccaaagtg ctgggactac agatgcacac caccacaccc ggttaatttt 15660 tgtattttta gtaaggacgg gggttcccca tgttggccag gctggtctca aactcctgac 15720 ctcaagtgat tcacctgcct tggcctccca aagtgctggc attacaggcc tccgccaccg 15780 cacccagccc aacctgggtc cttttgtatg tgagagtttg cttgtttttt tcacgtgctt 15840 tctctactcc agttttattc tatgacaaaa ttgaggccca acatgattta cttgcctgga 15900 tccacccaac ctgtcagtta cttcccagtg ctgctgccaa cttaatgtct ccttaaaagg 15960 atgctttaga gaaaacgaaa tcatgttgtt tttccccttt ggttaagaga tcaaacgccc 16020 accaaaagcc cttgggtcag tttcttagta gataaaaata attcttcgtc actttctgaa 16080 agcggctaac atataaccct tatgatgaat aatgtggtgt gtgtgtgtgt gcgcgcccca 16140 aattccaatg agttatcaaa gccagaaact tatattttaa atatgtttat ttcccaacca 16200 cactggaaac cacacacaga aaaaaaaaaa agcatgatta taccccctta ataaccgtta 16260 ctgcagaagg atgtgactct ccttcaacac ttgttggtat tttacagcct ccaaatctga 16320 ccatgtataa ccacctggga tagagttatt ttatttcaga accataatac ttagctatct 16380 cggaagttgc caatataaaa tgtttactct ctaatggttt tgaactaact caagacctgg 16440 ttatcccggg gagcatcctt acaaatgatc tgagagctaa cagtcctctt gcagcagtgg 16500 agggaaacac tcccgtggca atcactctcc aaaagccaga atgtgcaaga taaaagggca 16560 ccttccctgc agggaggcac attaagtcag tctgtgatct gctgccaaca tcctgactgg 16620 agccgtttct acgcctaact aatcatgacg tttgtgaatt gtgaagcttg ttgcaattca 16680 caattaactg ttaattgacc catattttat aacccgccag ccatgaactt acaagttaga 16740 tacagacact accagacatt cactattttt ttttacaatt gttttaaatg acattaatga 16800 gcatgcttga ttcctgaact cttctttaca gtataatttt aaaatatttg agtgggatac 16860 gatggagagg agggaggtgg gggaagaaat gccccatgga aaacccactc atcaggttga 16920 gagtgtggag aagccctgtg tatctgagaa ctcttaatca tccacagaca tggtatctct 16980 caaagagaag tgggtgtaat tccaaaatct aattttggca ggcgctcctg actaaatact 17040 taatctggag atgtcttcaa ggcaggcgga ggttttcagt cctggctgca cattagaagt 17100 cccaggggag ctttaaaaaa ttcccacgtc ctccctgcat cccagactaa ttaatcggga 17160 tctccgaggg tgggaccaca catcagggtt ttgtaaattt ccctgggggt ttggtggggt 17220 tgggggtgga ggcgtctatc ctatggccaa ggttgagaac cactgctttt taaaagactg 17280 tttgcttgtt tttgagatgg ggtctcgctc tgtcacccag gctggagtgc agtggcgcaa 17340 tctcagctca ctgcaacctc tgcctcctgg gctcaagcaa ttctcctgaa aaaggctgtt 17400 ggttattaat gcttccccac agctattcta ttcattgttg catgcttctt acgtgtgcta 17460 ggatgggagc tttaaaggat tacctcattt aatcctcaca accaccttgt gagagaggtg 17520 tcattatccc tgtttggaga gtgagacagg ggcttagcaa gctcagtaac ctgtccaagt 17580 cacacatctg catggggtta gctgctgcta aagctcatgc cgttaatctc catggtacac 17640 ggtgtcctct ccatagcaat cttgcggctg ccttgttaac accaaaaaaa cttgcatcag 17700 ctggtttgac aatttctaga taaagagctc ttttcgggct gctaagaagc ctaatttttc 17760 atttgatttt cttcttgaac tgtgtcacac tcctcattca tttgatatat tcatcaaata 17820 cttattgagc acctgctgtg tgcctggtgt gcagcagtga caccagacat ccaaagtcct 17880 tttcctctta gagcttattc tatctgggag agacagataa taaacacaaa atcagtaagt 17940 cattttatat ggtggtaggt gccttgagga agatgagcca ggttaatggg attaagcctg 18000 gtagggggag ggtgccactt tagctcggaa agggtagcga gacccaaaca atgcaaagga 18060 cccggcccgt ggagatctaa gacaggagga tgccagggac aggaagttgc tggggcaaag 18120 cccctgaggc tggactgagc tcagtgttct aggacgggcg tgggcagtga ggagcagcag 18180 aggaggtgag ctgggagata gcctggggac tctttcttct gcctccttca aaaaataaaa 18240 ctagccaggt gtggtggctc acacctgtaa tcccaacaat ttgggaagct gatgtaggtg 18300 gattgcttga gtccaggagt tcgagaccag cctgggcaac atagtgagac ccctcccccc 18360 atttctacca aaaaatcaaa aaattagctg ggcccggtgg cgtgcgcctg tggtcccagc 18420 tactcaggag gctgaggtgg gagcattgtt tgaacccggg aggtggaggc tgcagtgagg 18480 cgtgattgtg ccactgtact ctagcctggg tgacagagtg agactctgtc tctaaataaa 18540 taagtaaatc tagaacctaa catcttggag tgcagtggca ccaccatggc tcactgcagc 18600 ctcaatctcc tgagctaatc gagcctcccc ttcagcctcc tgagtagctg ggactatagg 18660 cgtgcaccac catacctgaa taatcaaaac ctaacatctt taaagaacat tggcataaga 18720 cttggcaaaa atggcatctt gtccctcatc tcatttagtc caagcgatac aggaaatgct 18780 gccacctcca ttttatagat gaggagtctg acgttcctag aggttcaatg ccctgaaacg 18840 tcaagccttg aggaagttgg agcactggga ttcgaagagc accatccaat acagacccag 18900 aatcaggatg atttgggatt atgcttgtca aggactcagg gcagggctac catacattag 18960 gcacaagaat tttgatagtg ataattactg tgttcattgt cacttcatca tgacagttac 19020 cgtgatgata agaaacctgg cccttcttca cctgacaaag gctttcttcg tttgagccac 19080 tgctcaaacg agactgacca agaataaatc ctcggggctt tggcctttaa aataggaagt 19140 catcataaat gacttgatgt ggtgtgtttc attcttgctt tgcaccagtg gaaaatatac 19200 aggtcaagca tcaaaacatg gcaaatgggg accccaatta ttagagaatc taagttaatt 19260 tttatgtata attaattatt caacaaccct ctcctctcca aaccaataat taatccatct 19320 tttgtatttt aagaccaatt ctgtagtatt ttccatcaat atctatttac tgctagcaga 19380 tatcagctac attctttctc ctttaataga agttccctct ttaggtatta agattcatta 19440 aacaacaata acaaatctac cttgcctccc agggacaatg cacagttctc attcatttgt 19500 tcatttagca gataattttt gaatttccac tgtacagcag ccctgtgctt gtggttggcc 19560 tgttatttga gaagcatcaa ataataatct cattttttgg ctgggtgtga tagctcacgc 19620 ctgtagtccc agcactttgg gaggctgagg cgggtggatc acttgaggat gggcgttgga 19680 gaccagcctg gctaacatgg tgaaacctcg tctctattaa aaatacaaaa attagccagg 19740 tgtggtggca gacacctgta atcccagcta ctcgggaggg tgaggcagga gaatcgcttg 19800 aacctgggag gcagaggttg cagtgagccg agatcgcccc attgcactcc agcctgggca 19860 acaagagcga gactccgtct caaaaaacaa aacaaaacaa gacaaaaaaa aacccaacaa 19920 ataaaataaa taatcccatt tttctccatt tttgagaaag atttctttgg tctgaagtct 19980 ttctctcccc tctccgaggc attacccagt ttaacctttc atgtataata tatatgatag 20040 ttatttaaag tatagcagga caaaatgtat ttgataggag aaaaccttgt ttgctctgtg 20100 ttaagtcctc cagagagcta attagagttt gtgattctaa aaggcaacta tagattcact 20160 tatattagca gttcatgtag attccagtta aggaaatggt ttgtcacttg tgttattgaa 20220 aacacacaca gggcgagcac tgtggcccat gctggtaatc ccagcgtttt gggaggctga 20280 ggtgggcaga tcacggggtc aggagtttga gatcagcctg gccaacatgg tgaaaacccg 20340 tctctactat aaatacaaaa aattagctgg cagtagtggc aggcgcctct aatctcagct 20400 actcgggagg ctgaggtagg agaatcgctt gaacccagga gtcggaggtt gcagtgagtc 20460 gagatcgcac cattgcactc cagcttgggc aacaagggca agactccgtc tcaaaaaaaa 20520 agaaagaaaa cacacacaca aaaaaacttt agtagatctt tcggcatatt attttttaaa 20580 ataaactgat aatggttgat atgattgttc aaagaaataa gagcttttca taaactcagt 20640 ttaaagaaac tttacaggcc gggcgcggtg gctcatgccc gtaatcctag cactttggga 20700 ggccaaggcg ggtggatcac ctgaggtcaa gagttcgaga ccagcctggc caacatggta 20760 aaagcctgtc tctattaaaa aatacaaaaa ttagccaggt gtgttggctg gcgcctgtaa 20820 tctcagcaac tcaggaggct gaagcaggag aatcgctgga acctggtagg cagaggttgc 20880 agtgagacaa aatcgtgcca ttgcactcca gccccagctg acaacagcga gactccatct 20940 caaataaata aataaataaa taaataaata aataaataaa ggagctttac agaaaccttc 21000 tgatgttttt ttcttcttga cgataacatt gccaacactg aatcttacaa agataagaca 21060 agaaagggac cttcagacac cattacatgt aattctggac ttagtggttt aaatccttat 21120 ttttctatga cattaaaaaa atgtatattt taggccaggc acagggctca cacctgtaat 21180 cccagcactt cgggaggccg aggcaggtgg attgcttcag cccaggagtt caagagcagc 21240 ctggggaaca tagtgagacc cctgtcccta cagatttttt ttttttgttt gagatggagt 21300 tttgctcatg ttgcctaggc tggagtgcag tggcacgatc tcggttcact gcaacctctg 21360 cctcctgggt tcaagcaatt ctcctgcctc agcctcccaa gtagctggga ttacaggcat 21420 gtgccaccac acccggctaa ttttgtattt ttggcagaga ctgggtttct ccatgttggt 21480 caggctggtc ttgaactccc aacctcaggt gatctgcctc cctcagcctc ccaaagtact 21540 gggattacag gcgtgagcca ccttgcccag cctacaaaaa gttttaaaaa attaaaaaat 21600 tagttgggca tggaggtgca tgccagctac tcgggaggct gaggcaggag gattgcttga 21660 gcccatgaag tggaggctgc agtgagccat aattgcagca ctgcactcca gcctgggcca 21720 tagagcaaga ccctgtctca aaaatatata tagtatccaa ataaacacaa taattacaga 21780 aaattgaaaa gtgcccataa gcaaaaaaaa aaaaaagaaa aaattaatca cctgcgttct 21840 catcacccag aattaaccat tgttaatatt tttgttatag atccttccaa acttttctcc 21900 atgcttgtga ttgtatttat tatacatgat ttacagggat ataaacgact gtattattag 21960 tcattagaag aactggatta tggccgggca cggtggctca cacctgtaat ctcagtactc 22020 tgggaggctg aagtgagcag atcatgaggt caggaaatcg agaccatcct ggctaacaga 22080 gtgaaacccc gtctctacta aaaatacaaa aaattacctg ggcgtggtgg caggcgcctg 22140 tagtcccagc tactcgggag gctcaggcag gagcagagat acctatctgt tctcaggatt 22200 ttaaggtgtt gcgcggaaat aagaaaaccg tacagtgttt ctcactacaa agcagggtca 22260 ggagatgcaa acaaactgat gtgggggttc caagtgaggt ggaattccag acaggggccg 22320 ggaagacttc gtggaaaggg agaatctgag gtgggttttc taggatgggt aaagttcatt 22380 agaggaagag aagtgcaaca gaggaagttc ggtgagaggt agagggaagg cgttctgatc 22440 atgaaggaaa cactagaaaa ggtatggaga tagaaaaaga taaggcctga ttttttaacc 22500 taccacttaa aaaaaatcct tgaaaagaga tttttaaaac gaatacttgg tgctgacaaa 22560 ggtgaaatga ccgggcgcgg tggctcacac ctgtaatctc agcacattgg gaggctgagg 22620 cgggcagatc acttgagctc aggagtttga gaccagcgtg gccaacatgg caaaactcca 22680 tctctactaa aaatataaaa attagacggg tgtgatggtg ggtgcctgta gtcccaacta 22740 ctcaggaggc tgaggcagga gaattgcttg aacccgagag gcggaggttg ctgtgagctg 22800 agattgtgcc actgcactcc agcctggata gcaggatgag actgtctcaa aaaaagaaag 22860 aaaaggaaag aaaaaaaaat ccgtactgta aactggtaaa ggctttcttt ctggagagca 22920 atttggggca catgcaccag tagccttaga aggctcatgc ttttgaccta attatcctat 22980 tagtggtgag atgattaaag atgtggcccc aatttatgtg aaaggtatgc atcacatctt 23040 cactcataat caggagagtt ggggaaaacc ctagctgtta atagtttatc caaaatccat 23100 atatatatgt gtgtgtgtgt gtgtgtgtgt gtgtgtatgg atttatatat atatataaat 23160 ggatatatat atatatctgg atggatatat aaatatgata tatatatgtg tgtgtgtgta 23220 tatatatatg tgtatatatg tatatatata tgatggaata ctatttagcc ataaaaagga 23280 atgaattaat ggcattcgca gtaacctgga tggacttgga gaccattatt attttatttt 23340 atttatttat ttttgagacg gagtctcgct ctgtcaccca ggctggagtg cagtggctcg 23400 agctcagctc actgcaagct ccacctcccg agttgacgcc attctcctgc ctcagcctcc 23460 tgagtagctg ggactgcagg cgcccgccat cacgcccaga taactttttg tatttttagt 23520 agagactggg tttcaccgtg ttagccggga tggtctccat ctgctgacct catgatccac 23580 ccgcctcggc ctcccaaagt gctgggatta caggcgtgag ccaccgcgcc cagcgagact 23640 gttattctaa gtgaagtaac tcaggaatgg aaaaccaaac atcgtatgtt ctcactcata 23700 agtgggagtt atgctatgag gacgcaaagg cataagaatg atacgataga ctttggggac 23760 tcagggaaaa ggtgggaagg gggtgaagga taaaagatac aaattgggtg cagtgtatac 23820 tgctcgggtg atgggtgcac caaaatctca taaatcacca ctaatgaact tactcatgta 23880 accaaatacc acctgttcct caataaacca tggaaattaa aaaagaaaaa agaaaaagta 23940 ccctggaaaa aaaatttctc cctggccagt cacggtggct catacctgta atcccagcaa 24000 ttcgagaggc tgaggcagga ggatcacttg agcccagtag ttcaaaacca gccagtgcaa 24060 catagtggga ccctgtctca aataaaatct aaaaattagc caggtgtgtt ggtgcatgtc 24120 tgtggtccca gctactcagg aggctgaggt gagagtattg cttgagccta ggaggttaag 24180 gcggcagtga gccgtgattg tgccactgcc atccaacctg ggcaacaaag caagaccctg 24240 tctcaaaaaa aaagaaaaaa aaaacctctc tattcgcctt ttaagaatac ctgggcttct 24300 ctgtgtacac ttaagcttca ttggagtctt tagacttttt ttttgctgta tctgtccagt 24360 taccaagtcc cagcttctac tccatgctcc ccatgctctc ttcctatttt attttccatg 24420 actgcctcgg tataacttgt gctcaaccaa actggactac tcaattccct gcattttctt 24480 ttttaaagtt taatcaaaaa aaaaaagaaa actggctggg cacagtgggc ttctgcccac 24540 aatctcggtg ctttgggaaa ctgaggcagg aggattgctt aaggccaaga gttcaagacc 24600 agcctgggta acatagcaag acctccatct ccacaaaaaa atttaaaaat tgactgagtg 24660 tgatggtgtg cacctagtcc cagctgcttg ggaggctgag gcaggagaat tgcttgagcc 24720 caggagttcc aggttatgat gagctatgac tgtgccaccg cactccagcc agggtaacag 24780 agtgggactg tctcaaaaaa caaaacaaaa tccctaatat aatctcagtg tgccttttaa 24840 gtatgccata tatatatata tatatatata tatatatata tatatatatc acattttctt 24900 tatccactca ttgattttca tgtagttcta atcgtagaat tcatacattc tttctatctt 24960 ccatctttca cataacatca caaacatttt ctaggttgcc atattgtctt catagttact 25020 taaataatat tccatcaagt agcacaatca tttatttcac tagtcctcta actgtagaca 25080 ttttggttgt ttttgaaact taataatgta aataacaccg tgataacaat gtttatgtaa 25140 attcatattt tggattatct ccttagggtg gattcccaga agtcacatta gtaggtcaaa 25200 gagtatgagc ctattttcaa ggctcttgtt ttattacctt ttaatttcca cttgcctcaa 25260 tattgctggt ttgctccctt atgatcacca gagttactcc gtcggtccaa attctttacc 25320 ttccgaaact gggaaggcca tgactcaatg ttatatatat agtaaaggct actataacct 25380 tccccagaat tttccaagcc agtggtctct aaagtgacct ttggctgtta aaatctgaat 25440 tcagagggtt catgagactc agtgttgttg tagaatttaa gctccttaat ttgccacgtt 25500 gtttagacac cacttaatac tttattgcaa atgacttgtc aacgcctctc acctacaaac 25560 ttcatcctcc tacaaatata cctcctgcta atcaaatgag gctacagttg agtctttaag 25620 tttcagtaga aagatggccc ttcctctggg gtaggcgcat gctcttcatg ctgaagctca 25680 gctgaaaagc ctcctgctga gttttctgcc tctttccctc ccactgcaca caccccaggg 25740 tgttggcgcc acttcaaagg gagcctgtgg atgaagaaaa cacaggtaaa ggcagagggc 25800 tcataagggg gccataaatt taaaaagtta agattcctgg cactatcaac tctcacttgt 25860 tttcaaatat gcatatggag tggatattcc agttttcatg tctgtgttgt tgtttttaaa 25920 aaaagacctt tcaaagaact gtgcattttt tacaggctga caggctgtgt ttggtgttaa 25980 actgtcaggg ctgactggtc acttggaaag ggcaagggct gaggtgcatg caagtgtcgg 26040 ctggttactc acagacacag cagccccttt taccccggag agagttctgt ttgctggagc 26100 ccttattctg gccagcagtg tcacaaatgc acactgtaag acatagacag tcttggaaag 26160 aaagggaaac tggctttaaa aattcttact ccttctagca aagcaattca tctttggcta 26220 taaagaataa cacagccagg tgcggtggct catgcttgta atcccagcac tttgggaggt 26280 caaggtgggc agatcacttg agtctaggag ttcaagacca gcctgggaaa catggtgaaa 26340 ccccacctct accaaaaaaa aaaaaaagaa agaaagaaaa gattagccag gtttggtggt 26400 acgtgcctgt agtcccaggt actcgggaag ctgaggtggg aggatcgctt gagcctggag 26460 ggcggaggtt gcagtgagcc gagatcatgc cactgcactc cagcctgggc aacagagtga 26520 caccctgtat caaaaaaaaa aaaaaaaaag aacagtaaca cattattaga aatgagcatt 26580 ctgaggccag gcacggtggc tcatgcctat aatcgcagca ctttgggagg ccgaggcggg 26640 tggatcacaa ggtcaggaga tcgagaccat cctggctaac acggtgaaac cccgtcttta 26700 ctaaaaacac aaaaaattag ccgggtgcag tggcgggtgc ctatagtccc agctactcag 26760 gaggctgagg caggagaatg gcgtgaaccc ctgggaggcg gagcttgcag tgagccgaga 26820 tagtgccact gcactccagc ctgggcgaaa gagcgagact ccatctcaaa aaaaaaaaaa 26880 aaaagaaaga aacgagcatt ctgaaatagt cttccatatg atgcttttga caattcagca 26940 ggaaaataaa ggatgtaaga aatgaatgca tatgttaggc ctcttgttga cctgtggact 27000 aaattgtttc tccctgcaga gatcagcaag gacaactcct gcaaagaaaa ctgtacttgt 27060 tcctcctgct tgctccgggc ccccaccata agtgacttgc tcaatgatca ggacttacta 27120 gacgtgatca ggataaagct ggatccgtgt cacccaacgg tgaaaaactg gaggaatttt 27180 gcaagcaaat gggggatgtc ctatgacgaa ttgtgcttcc tggagcagag gccacagagc 27240 cccaccttgg agttcttgct ccggaacagt cagaggacgg tgggccagct gatggagctc 27300 tgcaggctct accacagggc cgacgtggag aaggttctgc gcaggtgggt ggacgaggag 27360 tggcccaagc gggagcgtgg agacccctcc aggcacttct agagctcttc ttcttccttc 27420 attggcctct ccggatgttg aaacaaccac aggtcaagaa ggaatgtgaa tctgttgttt 27480 tataagagtt taggacaagg acgtggaaca gtggacactg gttttcccca aagctggcag 27540 ttttgtggag gggtagcttg tttcggtggt ggatctctgt ttatttttgc acatctgtta 27600 taatttaata ttcaaatctg gaattaagaa aacatatttt ctagtatcct ctaagggcca 27660 aagtcctaca atcggaatgg attcatgcca cgttgaagat aaaattatcc tctctctgaa 27720 atacggtaaa gatttaaata ggtcctgaga ctgttgatag ccccagacat acccacagca 27780 ttatatgtaa catctctcct gatcagtgcc attcccacgg tttcaaagaa aacagctaca 27840 aggaatgctt acctgagtgt ctgcagcacc ctccacttct ctcctaggca atgagaccca 27900 gtggctagaa attcaccatg tctattctca agatccatgc cagggagctc tttgactctc 27960 gtgggaatcc cactgttgag gttgatctct tcacctcaga aggtctcttc agagctgctg 28020 tgcccagtgg tgcttcaact ggtatctatg aggtcctaga gctccaggac aatgataaga 28080 ctcgctatat ggggaagggt gtctcaaagc ctgttgagcc catcaataaa actattgcac 28140 ctgtcctggt tagcaagaaa ctgaacgtca cagaacaaga gaagattgac aaacttatga 28200 tagagatgga tggaacagaa aataaatcta aatttggtgc aaatgccatt ctgggagtgt 28260 ccctcgctgc ctgcaaagct agtgctgttg agaagggggt tcccctgtac caccacatcg 28320 ccgacttgtc tggcaactcc aaagtcatct tgccagtccc ggtgttcaat gtcatcaatg 28380 gcagttctca tgctgtcacc aagctggcca tgcaggagtt catggtcctc ccagtcggtg 28440 cagcaaactt cagggaagcc atgcccattg gagcggaggt ttaccacagc ctgaagaatg 28500 tcatcaagga gaaatatggg aaagatgcca ccggtgtggg ggatggaggc gcgtttgctc 28560 ccaacatcct ggagaataaa gaaggcctgg agctgctgaa gactgcgatt gggaaagctg 28620 gctacactga taaggtgatc gtcagcatgg acgtagaggc ctccgagttc ttcaggtctg 28680 gaaagtatga cctggaattc aagtttctcg acgaccccac caggtacatc tcacctgact 28740 gtctggctga cctgtacaag tccttcatca aaaactaccc agtggtgtct actgaagatc 28800 cctttgacca ggatgactgg ggagcttggc agaagttcac ggccagtgca ggaatccagg 28860 tagtggagga tgatctcaga gtgaccaacc caaagaggac agcctcggcc gtgaatgaga 28920 agaagtgcaa ctgcctcctg ctcaaagtga accagattcg ctctgtgact gagtcccttc 28980 aggcgtgcaa gctggcccag gccaatggtt ggtgtgtcat ggtgcctcat cattctgggg 29040 agactgaaaa taccttcatc actgacctgg tggtggggct gtgacctggg cagctcaaga 29100 ctggtgcccc ttgctgatct gagcgcttgg ccaagtacaa ccagctcctc agaattgaag 29160 aggagctggg cagcaaggct aagtttgccg gcaggaactt cagnaacccc ccagccaagt 29220 aagctgtggg caggcaagcc cttcagtcac ctggtggcta attagacccc tccccttgtg 29280 tcaactccgg cagctcaaga cccccgagca acatttgtag gggccgctgc tagttagcta 29340 cccttgccca ccgccgtgga gttcgcacct cttccttaga acttctacag aagcaggttg 29400 cagtgagccg agattgcgcc actgcacacc agtttggaga cagagtgaga gtccgtccca 29460 gaaaaaaaaa aaaaaaaaaa gaacttntac agaagccaag ctccctggag ccctgttggc 29520 agctctagcc ttgcagtcat gtaattggcc caaatcaccg gagccacgtg accctccagt 29580 gtcatctccg gggtggccac aggcaagatc cccagtgatt ttgtgctcaa aataaaaagc 29640 ctcattgacc catgagaaaa aagaaaacag caatgagaag tgaccctgtc ttgttggttt 29700 attacttttt ttgttataaa gtactttggt gaattaacag gatgctagta ttacatggtg 29760 atactcttca gaacacctgc cccatctttt ttatgcaagt atgtttacaa tcagtggact 29820 atcagtaatg tcatttgctc aaatattttt taaagaccta cagaaactga tggttattgg 29880 gaaaacagtc aggaagtagt gaggtaatca aggccatggg aatagtgttt gacaaagaga 29940 gtactccaaa tcccttttgg ttacccagga ctttaaaaaa gagagtactc catcacacct 30000 gtaatcccag cactttggga ggccgaggcg ggtggatcac gaggtcagga gatcgagacc 30060 atcatagcta acatggtgaa accccgtctc tactaaaaat acaaaacatt agccgggtgt 30120 ggtggcgggc gcctgtagtc ccatctactc aggaggctga ggcaggagga tggcttgaac 30180 ccaggaggcg gacttgcagt gagccgagat agcaccactg cactccagcc tgggcgacag 30240 agcaagactg tgtctcaaaa aaaaaaaaaa aagagtgctc caaatctcct ttggttaccc 30300 gggactttaa aaaatttaat gtgatagtta ggccgggtgt ggttctcacg cctgtaatcc 30360 tagcactttt ggaagctgag gcgggtggat catttgaggt caggagttgg agaccagcct 30420 ggccaacatg gcgaaacccc gtctctacta aaaatacaaa aattagccag gcgtggtggt 30480 gggcgcctgt aatcccagct cctcgggaaa ttgaggcact agaattgctt gaacccagga 30540 ggtggaggtt gcagtgagcc gagattgcgc cactgcactc cagcctaggc aacagagcga 30600 ggttccatct caaaaaaaaa aattgtaata ataataataa caatgtaata tttacttttt 30660 catcctttat ataaggctga gtgcttcacc cctgagatga agctcagtta agaaataaat 30720 gaaaatcccg taacctattg gtgaaaggta accaccccca gctcctacta gcccaactta 30780 aaacaggacc ccatcacact acacagcagt ttagccaaga aaagggggtc tttatgtgga 30840 cactgggagg gaagggattc cttcaaatcc aaactttaaa ggattttaaa caaatgaaac 30900 atttggttca aagaatagct gatgttttta tttgatgatt ttggagaaag gaaagtgtgg 30960 ggcataatgg ggtttgttat tggaaagatc agattttcta ggtaatttgg gtggagaaag 31020 acaaaaggca aagctttgac tgacaattcc atgaaagtgc tatttggttt tggttatggg 31080 cttagaaaat taagacactt agttcaattt ggaaggattc tgtataagtc cctgattaaa 31140 ataagcaaaa atgatgaata acactgattc agtgcaaccg aaagattagg attaactcaa 31200 aagaaagtta ttttctaaac caccgtgatt ttttccactg acaattacag cggttttcat 31260 taggttgctg acacatgaag tcagcctcac catcagttgc aaactctaaa ctagcaaaat 31320 ctattacaga gacatactta tcacttctga tttagtgcta atctcaccca gctcatcttc 31380 tcttgtcaga tttatgagat aaatgtcaga tttatcacca gatatattga aagtaacagc 31440 cagtaataaa atgtgagatt ttaaaaaata gattctttgg caaattggtg ttcagtgagg 31500 caattattaa acatttttgt cagccaggtt ccaggcactg tacagaagct gttaggagtt 31560 ctcaccatct acgaatttga tttgatgtat tgtattctca ttaagctatg tgtgacacat 31620 tgtcatttat tagcccagaa tttaaaaagc tgtggttgtt tagtgttggt ggtagcagac 31680 cccagcagtc tgatggtctg cactccttcc atcctgccac cccctgggga tgcaaagact 31740 ggatctcagg gtgacaatct tcttgcgcac gactgcctgg ccaagtgcct ccagaaagcc 31800 ccttccttcc cccatttcca cccaggccca cttgtcacct cagcctaaca ccagcctgca 31860 cagtctacgg ccaccatcca ggcagtggga gagggaaagg ggaggagggt ggaagggaaa 31920 acccctttct atacctctcc tcagcctgct ctttcctcct cccacctctg agcctccgcc 31980 tcccccagac agagacagaa aagatggaag aacaggtggg acctccaccc ccaccccaag 32040 ccttcatccc ggtggagggg gatgggaaga tttctctcat ttcaagagac tcctccacct 32100 cagactgaca aaaggcagag gcctggcaag aagaaagggc accctgggga agaagggcat 32160 tgaaatagca cctgccgggc cgggcacggt ggctcacgcc tgtaatccca acactttggg 32220 aggccgaggc gcgtggatca cggggtcagg agttcaagac cagcctggcc aagatggtga 32280 aaccccgtct ctactaaaaa tacaaaaagt agccaggcgt ggtagcgggt gcctgtagtc 32340 ccagctactc cggaggctga gacagggaac tgcttgaact gggaaggtgg aggttgcagt 32400 gagccaagat cgtgccatgc actccagcct gggcgacaga gtgagactcc atctcaaaaa 32460 accaaaacag aaatagcacc tgcccccacc ccctgcccgc cctccttccc gcccccgtcc 32520 tttcctagac ttcactcaag tcctctgctc agaggaagcc ctgctctact gaaagccaca 32580 aggccattct cggtggcctg ggacagcagc ccaagacgtg ggcttctaac tgcctccgaa 32640 ggggccacag cagcaaacat aaataaaaat agtaaaatgt tcttaaatta taaatttaaa 32700 atttggaaaa tttagtgagc acagcttcta gggggcatgt ttccaaaatt ccaaccacaa 32760 aagtgcagtc tcaaaactga ctgtaaaccg aacataccat ctcatctcag acacagctat 32820 tgttcacgag tgtcagtgga actttcctcc cttgagatgg accaaaaacg tcaagcaaga 32880 tgacatttgc tgatttgcag gcttcaggca gataagatac gggcagagtt gagtgtgcgc 32940 ctttaccctt aaattcagga atagcaggaa cagcaggaaa aacgtaggac cacagcgtac 33000 gtcccacttg tctttcattt tgatatcatt atttccagag tcctgattgc tagtcatgtc 33060 taacactgga tttattatca tctcattgct agcatggcta ggaaagcttt gaacatcctt 33120 atcattctat tttaattcct attataattg catggggaag ttccagggtg gaaaaatttc 33180 cttttctttc tttttttttt tttaatagga gagttggctg ggcacggtgg ctcacgcctg 33240 tgatcccaac actttgggag gccaaggcgg gtggatcacc tgaggtcagg agttcgagac 33300 caacctggcc aacatagtga aaacctatct ctactaaaaa tacaaagtta gctgggtatg 33360 gtggtgcaca cctatagtcc cagctactgg ggaggctgag acaggagaat cacttgaata 33420 cgggaggcag cagtgagcca agatcatgcc actgcactcc aacctgggag agacagagtg 33480 ccatctcaaa aaaaacaaac aaaagagttg atataaattt gctgttataa tttgactgta 33540 ctgtttcttg cacatgttga catctgtaat gactggagtt tatgaaaatt tttgatgagt 33600 aggagcatac cattaacaga gagaaattta atcaaaagat ttttaaagtt ccttcagagt 33660 ccagactttg actaagtgta gtatgattta tatctatgtt gcatacaaaa atatcaaaca 33720 gtaattccca actgaaatac aagtatcaat caattgtgta acaatgcaaa atcatttaat 33780 ttaaagttaa tttatagcaa atgagtactg taatagcata agcatgccga tactttacaa 33840 aggagagagt ggaaaggtag gatattataa ctaattgatc aaatcattgt taaaatttaa 33900 gtttattaat acttttactt ctgtccgtag ggatccatgt taaattgggt atattataaa 33960 cttaactgct aatgatgagg tccttttgct attagaaatc tattttttat ttttctttat 34020 tattttttga ggcagggtct tgctctgttg cccaggctgg agtgcagtgg tgaaattata 34080 gctcactgca gcctcaacct cctgggctca aggaatcctc ctgcctcagc ctcccaagta 34140 atggaaactg cagtcgtata caagcacacc cagcaatttt tttttttttt tttggtaaga 34200 tggggtttag ctatgctgtc caagctggtc tcaaactcct ggcctcaagt gatcctccca 34260 ccttancctc caaagtgctg ggattacagg cgggagccac cattcccagc ctagaatgaa 34320 atatctttag ctaaattaca gggctggatg tggtggctca tgcctgtaat cccagcactt 34380 tgggagactg agggcgggag ggtcacttga gatcaggagt ttgagaccac cctgggcaac 34440 acagtgagaa ttctgtctct attttaaaaa gagaaaaatc tagggtatat tctcttaaac 34500 aaaactttca tctataatgg tagttgatga ggtcctatgt aatatgcatt tccttggttg 34560 caatagcaaa ttactacaca cacagaaagg aaagccacac tccccgacac atctacacac 34620 aggaggactc acacaggagg gagactcaaa gaaggcacgt gacttttaca ttgttagggc 34680 ttacatggtc ctgggatttc ccaccagtac tcaaaagatc aattgtatga acaagtcacc 34740 tatttttacg gcactaaata attattattc aacaacatgg aaaatatgtg gtagcagacc 34800 tggattttcc ttaagagtta tttttatgtg gtactgcccc ctgctggaat ataacatcta 34860 tacacatcct ttctggctgg gctgacatcc taaaaccagc ccaggaccag ccttttatta 34920 atattaattc ttggccaggc gcggtggctc gcctgtaatc ccagtacttt gggagtccag 34980 ggcgggcgga tcacgaggtc aggagttcaa gaccagcctg gccaacatgg tgaaactccg 35040 gctctactaa aaatacaaaa cttagctggg catagtggca cattcctgta atcccagtta 35100 ctcgagaggc tgaggcagga gaattgcctg aaccgggacc cgggaggtgg aggttgcggt 35160 gagccgaaat cgtgccactg cactccagcc tgggctacag agcgagactc cgtctcaaaa 35220 ataaataaat aaaaattaaa attaaaaaat aattcttggt tgtatgctaa aagccttgca 35280 agtagcccca ctggaagata ggaagagtgg ggctgtttta caaatgagca catataagca 35340 gaacgaggcc gccataattg aaatgaaggt ccccgtcccg tggatgtgtt catcgctact 35400 tcaccctgtc attcggatcc aatgtgtgac cagccagctc caataacagt tccatactct 35460 gggaattatt tttaacactc ggcaggatgc tttcttcctg tagttttagg cttagccctt 35520 tgtgcacttt tggtctcttt ccctttcaat ttagcatcca aggaagcggc tgtgaccaaa 35580 ggtagctgtc atgttaaagg acaaagttca tagttacagc aaatattgac ccagagcact 35640 atccttgccc cttcctctat aatgtgcaat gcaaaaatat gttcttttaa gtacaatatt 35700 aataagtaag gtctaggaga ttttcttccc ccttcctttc tcttttagat gagtaaatgt 35760 tttatctagt tttgaggaga ctatccttct tatcacatct ctttccactt ctgctctcct 35820 tgttttataa ttttcctctc ctttgggtcc gtgtcattat ttcgtgtcgc ttgttttcga 35880 gccatgcact catttatcaa atcagatttc ctccgtatgc cgacggcctt cctctccctg 35940 ccacgggctt cctttttccc tgactatgca gaagcaattt gttcgcttgt gtttcttttt 36000 ttttttgaga cagagtctcg ctctgtcacc caggctggag tgcagtggcg acatctcggc 36060 tcactgcaac ctccgcctgt caggttcaag caattctcat gcgtcagcct ccagagtagc 36120 tgggattaca ggtgtttgcc accaaggctg gctaattttt agtagagacg gggtttcacc 36180 atgttggcca ggcttgtctc gaactcccaa catcagttga tccacccatc tcggccttcc 36240 aaaatgctgg gattataggc atgaaccacc gcatctggcc ttgtctttca tccttaatga 36300 cactttagtc ctaataatgc taaaatcatt ttctactctt tgaattgaaa cacagcttat 36360 ctacatgagc ccaaggcagt agcaacattc acctccattt cttctctgat ctctaccttc 36420 tgaaccctgt ggacttggtt gtaaatggat gagggcaagt cttgcttcct tcccctgtgt 36480 ttacagagga tcgtggctga gatgctgggc cacactctgg gcctgctggc acccctgggc 36540 cggtggctgc tgcccctcag ggtgctcacc acctagacca gaagaaccaa ggtgagggag 36600 agcctgtttt ctttcttcct gtggctgcgg gggctgtgag gcatgggtct agtggctgtg 36660 tttagctggg gatgcctcct agaaatcagc tccaccgttg aagagatcaa agcaatgcac 36720 agtgccactt gaaatgaaac gattgagctt atcagcgctt ttgcaaatgt acaagagggt 36780 agctcccccg gacatcctga actgagccat gctcttctat tttgtgtaac agcccagtga 36840 cccctgaatc ttcccctgag gcaggtcccc gaagcttcat ggaggatgtt cctcagctga 36900 ccaaggtgag gctcttgagc tcctaaatct ttgtgatact gtttatacat ctttgtgctg 36960 tactttttaa gctgacttcg tgttatcacc tgtatgattt tatgttttgc ttctaaataa 37020 gtacagatta ttttaaactc taataatggg tgctacaaaa ttaaagatta tgtcaatcac 37080 tgtctctgat gagttatttt atgtagattt caacacaatc attgattcat gtgtactctt 37140 ggtcagtcat cagtcatctg agtacctagt gggtttccaa aatgggtcct ggatgctggg 37200 gatgcaaaga taagcaacac atttctatcc tcaacagcct gtagatgagg gagaatcact 37260 gcggacaatc agggaagtta ccggagagag cagtgcacat gtggtctaga aactggtgga 37320 acaaagttga gaatcactga actaggagga aagacaggtc actgacaatc caaggcacag 37380 tgactcacac tctaatctca gcactttggg aggccaaggc aggaagatcc actgagctca 37440 ggagataaag accagcctgg ctgacctatg gagacccttc tctaccaaaa aaaaaaaaaa 37500 aaaaaaaaac attagcctgg catggtgggg tgtgcctgtg gtaccagcta ttcaggaggc 37560 tgaagtggga ggatcgcttg agcccgggag gtcaagactg cagtgaatca tgatcacacc 37620 attgcactcc agcctaggga agagagcaag aaagaccctg tctcaaaaac agaaaaaaat 37680 ccagtaaaat gtttcagatg ttgttaaagg tgatttcact gttacttttc acctctcctc 37740 attttacatc tctgacctat gcttgtcctc tgacttgcca gacattccta gctatggact 37800 tgatgtctcg acatggaggc tcacaggcac cccaaactca gcctgcccta agctgaaccc 37860 atgatctttc cttccaaact tgtttctcac cagagttccc atcttatcat ccacctagtt 37920 gttcaagtca tccttaagac ctccctctcc ttcactgtct attctacctc cctaatatct 37980 cttaaatcct tccctcctct cccacctcac agccaccatc ctaacctaag cagccaccct 38040 ttctcaccct ataatgacct cctggctgtt ctctatagag ttggtgaatc ctttcgtctt 38100 cagcctgaac cccctttcga gggattctta tatatataca tagatataca caaatatata 38160 tgtacatatg tacatatgtg tgtatatatg tacatatgtg tatggatata catatgtaca 38220 tatgtggatg tacatatgta catgtgtatg ggtgtacata tgtacatgtg tatgggtgta 38280 catatgtaca tatgtgtatg ggtgtacata tgtacatatg tatatgggtg tacatatgta 38340 catgtgtgta tgggtgtaca tatgtacatg tgtgtatggg tgtacatatg tacatgtgtg 38400 tatatatgta catgtgtgta tgtacgcatg tacatatgtg catgtatgtg catgtgtatg 38460 tgtgtgtatg tacacgtgtg catatgtgtg tatatgtgta cacgtgtacg tgtgtatata 38520 tatatatata tatactggct ggagtgcagt gggaaagttt tggctcacca caaactccac 38580 ctcccaggtt caagtgattc tcctgcctca gtctcctgag tagctgggat tacaggcgtg 38640 caccaccatg cccagctaat ttttgtattt ttagtagaga cggggtttca ccatgttggc 38700 caggctggtc ttgaactcct gacctcaggt gatccaccca cctcggcctc ccaaagtgct 38760 gggattacag gcgtgagcca ccgtgcctgg ccggattcct atcttgaaga cgaagcccca 38820 gaccatcgac acggcctcaa ggccctgcat gacgcctcct gccccaacac ctcgtgtcat 38880 cttgctctcc tctcccgcag ctcctgaggc tttagccacc ctggaattcc aagtccccat 38940 ggtcattttt ttttcctgct caagatatca ccatgtgctg tcccctctgc ccttgtctac 39000 acccacgtgt ccttctcccg ccccggccac actcatgggg cacactgtcc ttccctggct 39060 aatcctccca cactcgatac cactttctct gggatattgc acccgatcct cagccgcagt 39120 tgtcttccta tgacccactc ccacactctc gccacaatgg taattgtttg attcctactt 39180 gttgtccctg tgagactgca aaccccagag gacaggggcc ctgggttctc cttcgcctct 39240 ggatcatcag cactaactga atacctggcc tagaagagat gctaacgatg ctgaatgaat 39300 aaataagtgg aaagactctc agtaaagcaa aacctttctt taccatttta tggccgtcaa 39360 ggaggaaaac acattatcag tggaaaacgc aaaatgaggg gatttgctta gcaaacgatg 39420 aattcctctg gcaccctggc agccttggtt tcttttgatg aggtccaccc ccttccatcc 39480 atcttctggg cttaagagat caaagcaaaa catgctgtgg aattcgatac tggtgcaggt 39540 tgcacaacat tgtgactgaa ctaaaagcca ctggatggta caactgaaaa tggtgaattg 39600 cgtgttgcat gaattataac ctaactgggg aaaaaaaggc ttaaaaagag acaaagcttc 39660 ccccacaatg gaaaggaagg tataatagaa acagcagctt tcaaaccttg gcaggataat 39720 gaaaccccgt ttctattttt aaaaattagc tgggtacagt ggcacgtgcc tgtagaccca 39780 gctactcggg aggctgaggc tggaagatcg cttgagccca ggagttcaag gctgtggtaa 39840 actataatca cactactgca ctccagcctg ggtgacagag aaagaccctg tctcaaaaaa 39900 ggaaggaaag aaggaaggaa ggaaggaagg agggagggag ggagggaagg agggaaggaa 39960 ggaaggaagg aaggaaatag cagctctgag cttagaaata ggagtctatt tctaagtggg 40020 agatggggag aaggagggaa ctggggaggt gaggaagaag caggtattgt caccagtgag 40080 gactgtgctg ttgtgagccc agctaggcaa ctggcaattc cattctgtta gtgacagcta 40140 caataaccca aagccctctg gagccctgct ttcctctgct ctcttcgtgg cttgactagg 40200 agctgaagat cctgtccctc ttagagcatt ggggcggccc accccacctc caccctcctc 40260 cacctgctgc ctcgaggccc ctcccactcc cggggtagac aaaacagttt agaggctgaa 40320 gtcaccgggg ctgtaactgt tggatttgca catgtcatag aaaatcatca tatgttttgt 40380 gtggactcca tgcataacaa caagagaacc aaccagaccc catagacaga agggagtgtg 40440 aattggagac aaaatttaaa ttatgagttg ccttctattc agatttctcc catttttaac 40500 aaaaaggagc ccaaattcct aaatgttatg gttgtttgca gcaacttatc atctttctcc 40560 tttccttcat agccaaggtt tttgaaagag ctatctgagg ccgggaatgg tgactcacgc 40620 ctgtaatcct agcacagagg ctgaggtgag tggatcacct gaggtcagga gttcaagacc 40680 accctggcca acatggcgaa atcccgtctc tactaaaaat acaaaaatta gccgggcatg 40740 gtggcgtgtg cctgtaatcc cagctactca ggaggctgag gcaggagaat cacttgaacc 40800 caggaggtgg aggttgcagt gagccaagat ctcaccactg cactccagcc tgggcaacag 40860 agtgagactc catctaaaac aaaaaaaaaa gtagctgtct gttctttctt ctcgaactct 40920 ttttcccgct ggagtctgtg acctgctgcc gtctgcctca agtgagaggg actagcagat 40980 ctggtgaatt accttctaat gcccgtaccc tgcccatacc agcttcaatc tgtatgtaga 41040 agcttagctt gctccatgca tggcctccag catccactgg tcacaaaata acacaaaata 41100 gcatgagaga gaatggtcgc atggagcgga ggagctgctg agactgaacc caagccaggg 41160 ctactgctgg gtggaactgg acatgcccag cccatgggaa agtcttccca cagaagtcat 41220 atttgcaggg gtctcccagg agacagcaca ttctgagcaa aggagtgagg cagagataac 41280 tattcaggaa ccaagagact cgctggaaag aagcagagat tttcagccca gcgtagtgga 41340 tgtttcttga atcttcccct gtggatgccc caaaccttga gatccttcca acaaatagca 41400 cactactaac aaactgtgac tcaaagagag ggaaacatgg tcccctgctc tgtcacaaat 41460 cactgtgaag ctttggcacc ctgactgctc aggtggccac caacacagaa ggaccacgaa 41520 tggctgagtc aggaagtcac agccgtgtgg ctggaagagg ctctgccttg ctctgggaga 41580 aatgcctatc cccaaggaag ccttagtatc catgggagag aaacactgta gcaatggccc 41640 ccaggactct cgggaagcca cttctggtgg gaggggactc aaagggtgct gggggacctg 41700 tgtctgcatc tggaagtgag gagccaggaa aattttcttt cagtttcttt cttttttctt 41760 ttcttttttt tttttttttt ttgagaaagg gccttgccct gtcgctcagg ctgaaacata 41820 gtggtgcgat ctcggctcac tgcaacctcc acctcccagg ttcgagtgat tctcctgcct 41880 cagcctcccg agtagctggg actacaggca tgcaccccca cccacgccca gctaattttt 41940 gtatttttgg tagagatgtg gtttcgccat gttggccagg ctggtctcga actcctggcc 42000 tcaagtgatc ctcccgatgt gctgggatta caggtgtgag ccaccacgcc cggcctcttt 42060 ctgcttcatt taacattaat ggtcatccca cagcatggtg ctgtgcacct gtagtcccag 42120 ctactcaggt ggctgaggtg ggagaatcac ttgcgttcca gctgtagtga gccttgattg 42180 tgtctgtgaa taaatgccac ttctctccag cttgagcaac atagggagac tgtctcttaa 42240 aaaacaaaac aaaacaggct gggctcggtg gcccacgcct acaatcccag cactttggga 42300 ggccaaggca agaggattgc ttgagcccag gaggtcaaga gcagcctggg caaaataggg 42360 agaccccatc tctacaaaaa gataaaaaat aaaaaaatta actgggcatg gtgatacacc 42420 tgtagtccca gctactctgg aggctgagat aggagtattg cttgagcctg ggaggtcgag 42480 gctgcagcga gccatgatca tgccactaca ctccagtcca ggcagcagag tgagatcccg 42540 cctcaaaaaa ataaaacaaa acaaaactca tctctccctt ggctcctgag actacaatcc 42600 ctcacggttc ttttctactt ctctgttttt ctcttcttgt ctcccttttt ttctggtctc 42660 tctgtcaccc aggctggagt gcagtggtgt gatcatagct cactgcaacc ttgacctcct 42720 gggttcaaga gatcctccca cctcagcctc tcgagtagct aggactacag gctcacacca 42780 ccatgcctag ctaatatttg tagattttgt agagatgggg tcttgctatg ctgtccaggc 42840 tggtctcaag ctcctggcct caagtgatcc acccacctca gccacccaaa gttctgggat 42900 tacaggggtg agccaccgcg cccagccgat aattgttgaa aaatcatttt cagttaaggt 42960 atccagtcaa ggtcagaaaa tgagaaaatg ttaaaaaaaa aaaagctata agtaaaacag 43020 attcagtcgg gacatgatgg ttcacgcctg caatcccagc actttgggag gttgaggtag 43080 gataatcact tgagcccagg agttcgagac cagcctgggc aacatagcga gaccttatcc 43140 atacaaaaaa atttaaaaaa tacccaggca tggtggcata ctcctgcatt ccctgctaat 43200 tggatgggtg aaggggagga tcccttgaaa taggagtaga ggtgcaggaa atatgattgt 43260 gccgtgtaat ccagcctggt tgacagagca agatgttccc cacccccctg aaaaaaaaaa 43320 aaaaacctaa atccaaattt taaaagtttc cttgactctt caacttgctc accctccacc 43380 aaataaaata actacgaagg aggcttattt tttactattt ccagggatac gatatatgtt 43440 tgtcctgaaa atatacatca tggctttact caagccacag tgatgaggcc tcattgtcac 43500 tgtagcctaa ttacgatttt ataactccat ttaaaattca atttaaacac agtttaaaaa 43560 ttcagtccaa gtcaaacatg ctctcagtag ctagaagcaa aactctgttc aggtccttga 43620 tggatctatt tgtactttct ttcatgaaaa cagaaagtcc ttttttacac accatgcaac 43680 aggaaaattc ataacggaca ttgttttacc tgttcttggc aaagacaagt gagctcttaa 43740 caagcaaggt aactatggag atgatgtttt gctccaagtt aacacttaca tatttaatta 43800 gaaagatttc aaaggtgggc agattcactg gaaagtttcc aaaagcttca cttgttcaac 43860 aaataatgtt agagagggag caccgtgccc tcgggcccct aggaattagt tccacatggt 43920 ccggtcctct gtccagtgtg cccagcatcc acttgggaga acagcatggc cttctgtcca 43980 gggcagccca cgccagcact gcctgccctt tcaggcccat ggctcccatt aagtgccatt 44040 tcgagcatac ttagccaagt ttccctacca tggccaacaa agaggttgtt caaaaatgct 44100 tgtcaggtcg ggcatggtgg ctcacgcctg tagtcccggc actttgggag gctgaggcgg 44160 gtggatcacc tgaggtcagg aattcaagac cagcctggcc gacatggtga aaccccgtct 44220 ccacaaaaat acaaacatta gttgggcatg atggcgggtg cctgtaatcc cagctgctca 44280 ggaggctgag acaggagaat tgcttgaacc cgggaggtga aggttgcact gagctgagat 44340 cacaccattg cactccagcc tgggcgacag agtgagaatc catctcgaaa aaaaaaaaag 44400 tttgtcaacg gtttcactga atccagaata cttttctaaa atgtcaaccc tatagaatac 44460 attttataaa attatgaagg cctggtctgg tgtagtggct cacgcttgta atcccagcac 44520 tttgggcagc caaggcaggt ggatcgcttg aggctgggag tttgagacta gcctggccaa 44580 caaggcaaaa ccctgactct actaaaaaat acaaaaatta actgggcgtg gtggtgcaca 44640 cctgtaatcc cagctactca ggaggttgag acaggagaat cacttgaacc caggaggtgg 44700 aggttgcagt gagtggagat tgcgccattg cactctagcc tgggtgacag agcaagactc 44760 tatcttcaaa aaatagataa ataaataaaa attaaaacaa aataaaattc tgaaggcctt 44820 aggtcagaga attaccgagg gaatattcaa agttatacct ccaagtatct acaatgaaga 44880 tactttcatc agaaaaaagg agtttacggc caggccctgt ggttcatgcc tataatctca 44940 gcactttggg aagccaaggc tgaggcagga ggatcacttg aggccaggag ttcgagacca 45000 gcctgagcaa aaacgtgaga tcccatttct accaaaaata aaaatgtaag gtaggcatgc 45060 aactgtagtc ccagctactc gagaggctga ggcaagagga tcgcttaaac ccaggactcc 45120 agcctgagca acagagcgag accctgttta taaaaaaaaa agaaaaaaaa aaagaagaag 45180 aagaaggaga agaaaggaaa taaaatttaa gaaaaaaaaa aggacttaat aaggttgaat 45240 gaaggcaaga atattcttag ctctgtttaa gtcaagacct gagtagtagc tctacgtagc 45300 tgtatgtcga taatgttttt gagacagcac tactgataaa ttgttacata ataaactgtt 45360 atggctggat gcagtggctc atgcccataa tcccagcacc ttgggaggcc gaagtgagtg 45420 gatcacctga ggtcaggagt tcgagactag cctgatcaat atggtgaaat cccatttcta 45480 ctaaaaaaat aaaaattagc tgggcatggt ggcgcacctg taatcccagc tactcaggag 45540 gctggggcag gaggattgct tgaacccagg agacagaggt tgcagtgagc cgagattgcg 45600 ccattgcact ccagcctaga agacagagcg agactccatc tcaaataaat aaactgttaa 45660 attaagttta gcctaaagct acccccttac atattttaag ttcagtctaa aggtttccct 45720 gcacatagtg aactgtaacc taactggatg cgtaaacaga ctataaccta ctcttgggcc 45780 agtcactgag ttttggtcaa tcaaaggcag ccaactgttc aaaccaggtt aaaataaggc 45840 agatgctgag ctctaaccag tccagccatt tctgtacctt gcttccattt tctgtccatc 45900 actttccctt ttctgtccat aaatcttcca ccacgtggct gtgctggagc cactgtgaaa 45960 ctattctgtt tcaggggctg cccaattcat gaatcattcc ttgctcaatt aaactctgtt 46020 catttaattt gtctaatatt tttcttttaa tcaaagtaat ttggccgggc acagtggctc 46080 acgcctgtaa tcccaacact tcgggaggcc gaggtaggtg gatcacctga ggtcaagggt 46140 tcaagactag cctggccaac atggtgaaac cccgtctcta ctaaaagtac aaaaattagc 46200 cgggtgtggt ggcgggcgcc tgtaatccca gctactcggg aggctgaggg aggagaatcg 46260 cttgaacccg ggaggtggaa gttgcagtga gctgagattg tgccattgca ctccagcttg 46320 ggcgacaggg caagactctg tctcaaaaaa aaaaaaaaaa ttaattcaga gacctactca 46380 tgtgaagttg tattttttta ttctccatat tacaaaacag aacaattggc acagggatga 46440 agaaatactt tgcaaaacat ctagagaggt taaatgccat gagtctttaa aatgtaagac 46500 tgctttcacc tgagcaatct agtgtccatt tctagagcta gcttaaatgt ccgtgtaaat 46560 ccccgtaatt ggttgggata acaattacct atgttgtata acttgagtca aaaactacgt 46620 ttccactgcc tgccacccct atggatggtt ttctcttaag gtatcaaatt ttactgggaa 46680 agacctagat aaaatacagc gaaaatgagg cggggcgtcc tggcacatgc ctgtaatccc 46740 agcgctttgg gaggctgagt cagaaagatc tttgaattca ggagttcaag accagcctgg 46800 gcaatatagt gaaatcctgt ctttacaaaa aattaaaaat tagccaggca tgggggcatg 46860 ggcctgtagt cccagctact tgggttgggt gactgatgtg ggaggatcac ttgagcccag 46920 gaggttgagg ctgcagtgag ctctgaccat gcccctgcac tccagcctgg gtgacagagc 46980 aagacccagt ctcaaaaaga aaagaaaaag agtaatgtta ggtcaaggta gaacctacct 47040 tgactttctg ttactatgga agatattctg gggtatctct gagatccaag tattatggca 47100 cttaagtaat tcctatctat tgttctactt ggttcctcgg gagtaaaagt catattcaaa 47160 ccaaaaaggc tgtgggattt ccagaatttt aaaagcaata atagttaatg ttctcccatg 47220 ggagttactc cacattttta catatgttcc atatgttaac tcatttagac cttaccttta 47280 tgaggtaagt cctcttctta tccccacttt agaggtggga aaactgaggc acagaaagag 47340 taagttgctt gcctaaggcc ctgttactag caggtggtga aaccagcatt ccaacccggg 47400 agtctggcaa atgtgtgtga agagcacacg tttggaaatg acagtcatga ggacactgta 47460 agacttctgg aatgtttata atttcacctt tgcttgttat ttttcctgtc tgtttcccta 47520 gagtgagctg agtgaaaaaa gaaagaagaa agaaagaaga aagagaaaga gaaagaaagg 47580 agagaaaaag aaaaaagaaa agaaaaacag aaaaagggaa agaaagaaga aatgaaagaa 47640 agaaaagaaa agaagaaaga aaagaaagag agaaaggaag gaaggaaggt gggaggggag 47700 gaaaggaaga agaaagaaag aagggggaca gagggaggga atgaaggagg gagagaggga 47760 gggaaggagg aagaaataaa aagatgagga tctgtatgct tgaggggtgg aggtgggggg 47820 cttgggtggg agtgtgggat gggcagaaag ctggagggag ccctggaccg actgcattcc 47880 acagaggatt gtgggtgcaa cgtaggtggc agattgagaa aagcaaacaa acaagctcag 47940 cctttggagc ttcggggaag aaaaaaagct gagcagtgaa tgctggcttc ccacggagaa 48000 ggcaggctgc ttcgccagct cacatccttc cgcgcaccca cttcctcttt ccggaggtca 48060 ctttagattg ctttatggca ggatctccag gtcacaggaa tgttatgttt cgactggggt 48120 ttccccctcc cctgggatgc ctgggccagc tccccaaggg ctagtctctg tcccaggccc 48180 cacactccca tagcactcag caaaagccta gagagagcac cgcaaaatgc caaacgcaac 48240 aggaccgcgt aggaagaaga cgcttggaat gacagggaca ctagaactgc ccatggtcgt 48300 ggtctcaaat ttttgttcca tggtctgaaa tactaaaagt tcttaaacag ctacttgatt 48360 tcatactatt gttttgaaga aaacagtgtt tgtttgttgt tttgtttgtt tgtttgtttg 48420 agacagagtt ttgctcttgt tgccgagttt ggtccatgtt ggtcaggctg gtctcgaact 48480 cctgacatca ggtaatccac ccacctctgc ctcccaaagt gctgggatta caggaaaaca 48540 gttgtttctt taaaacaatt atataggctg ggcacggtag ctcatgcctg taatcccagc 48600 actttgggag gctgaggtgg gtgaattacc tgaggtcagc agttcgagac cagcctggcc 48660 aacatggtga acctccgtct ctactaaaaa tgcaaaaaat tagccgggcg tggtggtgca 48720 ttcctgtaat accaggtact caggaggctg aggcaggaga atcacttgaa cccaggaggt 48780 ggaggttgca gtgagctgag atggcaccac tgcactccag cctgggcaac aagagcaaaa 48840 ctccatctca caatctcaaa aaaataaaat aaaataaaat aaaataaatg gttatataag 48900 ctaccttatt gatgcagtta caaatgagcc gctgaaacat ataaatttta aagaacaagc 48960 cacatatctt tcatcaccca cagcttcacc aactaaaggt gtatgtagta cttttgtgga 49020 aggcatttcc acatgctttg agggaccttg aaatactgct atgattacat gatttttcta 49080 aaaccagact actcctacat tacaagaatt gaaaagttca gagtaaatat ttgtaagacc 49140 tagaaaagat gatgttcttt aaaaaaaacg atgcccatct ttgtagcgaa aagaaagaga 49200 gatcagactg ttactgtgtc tatgtagaaa cagaagacat aagagactcc attttgaaaa 49260 agacctgtac tttaaacaat tgctttgctg agatgttgtt aatttgtagc tttgccccag 49320 ccactttgac ccaactactt tgacccaacc tggagctcac aaaaatatat gttgtatgaa 49380 atcaaggttt aagggatcta gggctgtgca ggacgtgcct tgttaacaaa atgtttgcaa 49440 gcagtatact tggtaaaagt catcgccatt ctctagtctc aataaaccag gggcacaagg 49500 cactgtggaa agccgcaggg acctctgccc tggaaagcgg ggtgttgtcc aaggtttctc 49560 cccatgtggt agtctgaaat atggcctcgt gggatgagaa agacctgacc atcccccagc 49620 ccaacacctg taaagggtct gtgccgaggt ggattagtca aagaggaaag cctcttgcag 49680 ttgagataga ggaaggccac tgtctcctgc ctgcccctgg gaactgaatg tcttggtata 49740 aaacccgatt gtacatttgt tcaattctga gataggagaa aaaccgccct atggcgggag 49800 gcgagacatg tttgcagcaa tgctgccttg ttattcttta ctccaccgag atgtttgggt 49860 ggagagaaac ataaatctgg cttacgtgca cgtccagtca tagtaacttc ccttgaactt 49920 aattatgacg tagattctgt tgctcacatg ttcgttgctg accttctcct tattatcacc 49980 ctgctctcct actacattcc tttttgctga aataacgaag ataataatca ataaaaactg 50040 agggaactca gagatggtgc cggtgcaggt ccttggtatg ctgagcgccg gttccctggg 50100 cccactgttg tttctctata ctttgtctct gtgttttatt tattttctca gtctctcgtc 50160 ccacctgact agaaatatcc acaggtgtgg aggggcaggc caccccttca catcttgtct 50220 ccacttcctt gattaaaaaa aagaaaagaa aaaaaaattt gccgaagttg gattcattca 50280 cagaattcta cacattaaaa atgttgcagg tcgggtgtgg tggcagctcc caaagctgcc 50340 tataatccca gcgctttggg aggcttgagc ccaggaggtc aaggctgcag tgaactgaga 50400 tcgcaccact gcactccagc ctgggcgaca gagcaagacc ctgtctcaaa gaaaaaaaaa 50460 aaaacagaaa aaaataacgt tacagaaaaa gtacaatatt tttaatatat atatatatat 50520 tttttttttc tgagacagag tgttgctctg tcacccaggc cggagagcta tggctcgatc 50580 tcagctcact gcaacctcca cctcccgggt tcaagcgatt ctcctgcctc agcctcccga 50640 gtagctggga ttacaggcac ccaccaccac gcctggctaa tttttgtatt tttagtagag 50700 acggggtttc cccatgttgg ccaggctggc ctcgaactcc tgactttatg atccgcctgc 50760 cttggcctcc caaagtgttg ggattacagg tgtgagccac catgcccagc caaaagtaca 50820 atatttttaa tgacatataa agatgttcat tctttgtggt tgccctgggt gagagggact 50880 attgatactc aatagtgttt cttttgtttc tacattgttt ctatagtgaa aatacgcatt 50940 ggctttgtat taaaaaatgt atagtaaaaa tggttttatt aaaaatagca aataactaca 51000 aaaactccat tgcaatggaa agcagccctt ggattttcta gttgaatgaa acgagtaatt 51060 tatccaatgt tagaaatgtc taaaggctcg ctcaggtttc atgagcagaa caggaattgt 51120 atatccaatt aaatgtgaaa ttgcaatgcc tggtgcggtg gcttatgcct gtaatcccag 51180 cactttggga agccgaggca ggggatcgct tgagcccagg agttcgagac caccatgggt 51240 aacatgggga ggccccatct ctacaaaaaa taaaaatcgt tagccgggca ggttggtgca 51300 tgcttgtgtt cccagctact tgggaggctg aggtggaagg atcctctgag cccaggagga 51360 tgaggctgca gtgagacatg atcgatgcac tccagcctgg atgacagagt gagaccctgt 51420 ctcaaaaaaa aaaaaaaaga aaagaaagta caatcgcaat taaatgtctt tgcgttggtg 51480 gctcctgacc aaattcccta agcaagcagt atgttaatga gcagaggggc cacagctcac 51540 cttgctcaat taaaggcagg agcaggccgg gcgtggtggc tcacgcctgt aatcccagca 51600 ctttgggagg ccaaggtggg cggatcacga ggtcaagaga tcgagaccat cctggccaac 51660 atggtgaaac cctgtctgta ctaaaaatac gaaaattaac tgggcatgtg gcatgagcct 51720 gtaatcccag ctactcggga ggctgaggca gaagaattgc ttgaacccgg gaggtggagg 51780 ttgcagtgag ccgagattgc accactgccc tccagcctgg tgacagagcg agacttcatc 51840 ttaaaaaaaa aaaaaaaaaa ggcaggagca agtatgggcc agacagaaat caaggtgtaa 51900 attgggcaga tcctcaggcc cagtgctgaa ttttggtttg atgaaataaa acattacatt 51960 tcaaggttgg cagagaggaa tgaaggtgga agaggaatct agggccattt agggaagcca 52020 tgaagcctcc tgcccacact agtgggtaga gtggagccag gcgttttgct agggcttgct 52080 atatctcttg gcagggtgct ctgctgccaa agccaagaat tctaaattag attaaatagc 52140 cagaaagaat gttaaacatt tggacatgat atcctccctc acagattagc tagagtgtag 52200 ttctgctgtg ctagatactt aaataaatac ctccctagct gtgaagcctg cttatcacag 52260 tactatattt taggatgagg tcattatttt cctatgcata cacatgcatt gtataatctt 52320 gccaatgtag gtcagcccaa aagaagtgac aaatgtgtag aacacacatt ggactagctt 52380 gggacaaaat tagtatacct aaagatgaca gatttcttaa ctaattttat gagccatgca 52440 gctttgtatt ctagcagaga cagacattag gaatcttata aaatcaaaaa ttttaatttt 52500 tgcctgaata gctccaaagg gctaagatct caagcaaatg cgtgtaggtt ttgtttttgt 52560 ggttgttgtt gtttttagag acagggtctt gctctgtcac ccatgctgaa gtgcagcggt 52620 gcagtcctag ctcactgcag ccttgacctc tcaggcttaa gtgatcctcc tgccttagcc 52680 tcccgagtag ctgggactac aggcgcatgc caccaccccg agtaattttt tatttttatt 52740 tttacttttg tagagacagg ggtctcaata tgttgctcag gctagtatct tttttctttt 52800 tgagacagtc tcgctcaatt gcccaggctg gagtgcagtg gtgccatctc ggctcactgc 52860 aagctccgcc tcccgggttc acgccattct cctgcctcag cctcccgagt agctgggact 52920 acaggcgccc gccaccatgc ccagctaatt ttttttgtat ttttagtaga gacggggttt 52980 caccgtgtta gccaggatgg tctcgatctt ctgacctcgt gatccacccg cctcagcctc 53040 ccaaagtgct gagattacag gcgtgagccc tcgcgcccgg cccagtcttg taacttaact 53100 ttaaagctac ttattcccaa atgaagatgg gatggtacac agattttaag tattagctgg 53160 tttggagctt ctgtctttta aagcaacatt ttactttgcc acagggtggt ggggcggggg 53220 ccatcctaga aagaagagtg tgagtttcat gggatagggt ctggggaggt ggctggagga 53280 gtttaggttc ttttgatatc tgtggctaca cagacagata accaaggaaa atgtccaaac 53340 agtgaaatta agtgctcact gcactaacac agagaaggac cctgatgtct ggccgcaggc 53400 ctttgttctc attggcttca aagaacttct tgatgtctac cttaatttca ttattattta 53460 cccaggagtc attcaggagc aggttgttca attgccatgt agttatgtgg ttttgagtga 53520 gtttcttaat actgagttct aatttgattg tgctgtggtc tgagacactg tttcgatttc 53580 agttcttttg catttgctga ggaatgtttc atttccaatt atgtggtcga ttttagagta 53640 agtgccacgt gacgctgaga agaatacata ttctgttgat ttcggggggg agagttctgt 53700 agatatctat taggtccact tgatccagag ctgagctcaa gtcttgaata tccttattca 53760 ttttctgtct cgttaatctg tctaatattg acagtggggt attcaagtct cccactatta 53820 ttgtgtggaa gtttaagtct ctgtgtaggt ctctaaaaac ttgttttatg aatctgggtg 53880 ctcctgtatt gggtgcatat gtatttagga gagttagctc ttcttgttga attgctccct 53940 ttaccattat gtaatgccca tctttgtctt ttttgatctt tgttgggata aaattacatt 54000 ttatgtcccc cttcctatag tttgtcactg agggttggca gaagttgaaa ggaagaagac 54060 atttgggtgt ttggtttggg gttatattag gttataaggt tcattgcctc cacctctttc 54120 aaaacattta gtttctaaat gaatccagct ttaaatgact gcaggagtgc ccatgcacaa 54180 ttttgtttct caaatctttg ggatttttcc ttgaagaata ttcacaggga atggggctgt 54240 cttgcttcat agttactctt ttgtatacat gatctcaaga atcgcctgat cactgctaga 54300 gttaaaccaa tacactaact gcctgaagtg ctgaaaagtc aaatgggggc ttagaacctc 54360 actccagatc ctacacaagc tgatggttct gttcccagaa acaacccagc ttcctcatca 54420 tctatggcca gtgccttgta gcggagctgg agatcaccct ttagtgggct cttcagctgg 54480 atctagaaat caaattgaca ccaggcagat taacaagaga aaagtataca gattttattg 54540 cttttatatg tacttgggaa tctgcacaag ggcaaagtcc gaagaggtgg ccaaagcaag 54600 gtgcttttat acatttttag aaaaagagcc aaaaaattgg agaagaaatg ataggacaaa 54660 gaaaatctag ccaggcagta aattttctag gagaatcact aggacatata tgaggaaggg 54720 tgtgtaaaac aggtgaaaga taagggctag ttcattaaac atgtttactc tggtccattg 54780 tagcctctac gataaggagt attttctcgc tctggtgtgg acagggcacg cctcccagag 54840 caacctttat cacttactgc atgcaggaag agacaggtca gcccgccctt cctgaaacta 54900 caatttcttc agtgttttca actcaaaata atcaataccc cccatctggc atatctgggg 54960 atggcacgtc ctttactcct tcaggctctt ctccctgaag gtcctttgca tagttgggaa 55020 tctccaccag gaggggtagc tctttggtct aaacccatgg tggcagagtt tcgacaatat 55080 tcccaactta aatgtttctg attctgagtg gtggttagat ccctttgtac acccctgtcc 55140 ccagtgccta cagaatgggc atgttaataa gtgttggctg aacattcaat gatggataag 55200 gaagaatagg aggcaagaga gacggtggtc tccagtgcca agccccagtg ctaactgggg 55260 tgattttttt tcatgactca ttttcctaaa atcaccctca agggtcctac aaaactcttc 55320 ccaacagcta aatcacagac taatctggcc catcgacgtc ttccctgatt atactaattt 55380 ttttgtgttt ttttttttga gatggagtct tgctctgtca cccaggttgg agtgcaatgg 55440 cactatctca gctcacagca acttccacct cctgggttca agcgattctc ctgcctcagc 55500 ctcctgagta gctgggactg ccagcatgcg ccaccatgcc cggctaattt tttttttttt 55560 ttttttagta gagatgagat ggggtttcac catgttggcc agggtggtct tgaactcctg 55620 accgcaagtg atccgcttgc ctcggcctcc caaagtgctg ggattacagg tgtgagccac 55680 tgcgcccgac catatattaa tggtttttga tgaatttgtt ccatagatta aaatcttgtg 55740 ccccatcgcg tgtggggctc catcgcatgt ggggcacagg gttcctgagt gtttgtggct 55800 gtcaaaccaa gatgatttct tgcttaatca agcagatttg aaagttcatc tctgctacca 55860 ggaagcactt gctcaactca gaagacaatg tcctatcagt ctttcactat cacgcatctg 55920 ttcttcaaga tccgtcaaat tagctccagt gaaacggagg ctaaagtgaa actttttctc 55980 ttatatagat ttttattcat aactagggaa aaattaggca cccacagaaa aataataacc 56040 taaaaaaatt aggctgaacg taagaaaaat ttgtgatgaa ataaacattt caatcaacag 56100 aaaatatttt tctgactttt tatgtgccac cattagttac atcattgaga aaacaatatt 56160 tgtattaaaa aaagagctgg tgaaaatctg gcaattggtc gggcatagtg gctcgtgcct 56220 gtaatcccag cactttggaa ggccgaggca ggcggatcac ttgaggtcag gagtttgagg 56280 ccagcctgac ccacgtggtg caaccccctc tcaactaaaa atacaaaaat tagctgggcg 56340 tggtggcagg cgcatgtaat cccagctact agggaggtta aggcaggaga attgcttgaa 56400 tctgggagat agaggttgca gtgagccgag actgagccac tgccttccag cctggtgaca 56460 gagcaagact tcatctctct ctcttttact ttttttaaag acttcttctc aaaaataaaa 56520 agaaagaaag aaaatctggc aatccagtaa aaactggcca ctatggcatg catgtgctat 56580 gcataaacgt aaattgatgc ataaacttaa ttttagaact ggaaggaaat ctggagttct 56640 ttaggagcca ggttttacac atgcagaaac ctaacagctt cagtttcgat tcgataaaat 56700 ttgactaact aaacttaaga taagcatagt tacgcattag agtattaact ctcaaacttt 56760 taaaaaagaa ttcttccttt gcttgttaat tttctttctt tctttttttt ttttttgaga 56820 tagggtcttg ctgtcgtcca ggccgaagtg cagtgacgtc atcatagttc actgcagcct 56880 ctacctcccc ggctcaagta atcctcctgc ctcagccttc tgagtatctg ggactacagg 56940 catgagccac catgcccagc cttttctttt cttttctttc ttttctttct ctctctgtct 57000 ctctttcttt cttttttcct ccctttcttc tttttttttg atggagtctt gcactatcgc 57060 caggctggaa tgcagtggtg cgatcttggc tcactgcaac ctccgcctcc gggttcaagc 57120 aattctcctg cctcagcctc ctgagtagct gagactacag gtgtgtgcca ccacgccagc 57180 taattttttt atttttctag agacggggtt ttaccatgtt ggccaggatg gtcttgatct 57240 cctgacctca ttctccacct gcctaggcct cccgaagtgc tgggattaca ggcatgagcc 57300 accgtacctg gccctttctt tctttttatc aagacaacaa catgtcttta tagtgctccc 57360 aaggctaaag tataccttac gtctatgtaa acactcaacc tgagctttgc aatggcccat 57420 gttggcagta gtgcaaacaa aaacaattat gaaacccatt ttcctttgac aaagagaaat 57480 aagtggcaag aattggttct ttctcttagt atgggtctct gaaaagaacc agatcagtca 57540 aaaggggaat atttttctga agggataggt ttggcctagt ggcttctacc tcttttagat 57600 gactgctgtt tctcgtttta atgttaaata gacactaata ggagaaatca cattaattca 57660 gtcaacaaac atttactgag cacttcctgt agtcaggccc tctgttaact tctgggaata 57720 caatgacaac tctgacaatc ccaacccaag gagccaacaa gtccgggaat agagacagac 57780 aagaaaacag acaattacaa ctctaccgtt agaataaagg tacattgaga acttgcaaca 57840 aatattcctc atcccttatc ttaattattc ataacatgtt taccaccaat aagaatagca 57900 ataacaataa atgcccaact cagacagcaa tgtccattta ccctgtgttt acacagcata 57960 atacaagcaa gctgtggaca gagattctct tgtttagtcc tcacaactct gcaaggtggg 58020 ttttattact ctccatttct agataaagga tctcacctaa tattacatgg gccagtggtc 58080 ttccagttgg ggtatgcaca accctagggg taggtgagga ccctgcctgg ggtcttcagg 58140 tggggaccat caacctccat ttgtactctt ttctgaacat tggtctgaga cagaaagtcc 58200 ctgcaattaa ggcattaagc tggctctttt tctatttctc atttcataat tgcccttctc 58260 ctgctttacc aaaatctttc accccccatc atatatatat atccccatac atattctata 58320 tatacatacc ctacatatgc atgcacacac atcatatata tgtatgcata tatgatatat 58380 acatatatgc tatgtaaaca tatatagtgt gtatatacat gtgtgtatgt gtatatgtgt 58440 gtatatgtgt atatatacac gtgtgtgtgt acatatatac atcatatatg tgtgtctata 58500 tatgtatata tgggtgtgtc tatatatgtg tatatgtacg caaatacgta tatgtgatgt 58560 atatatataa gatgtgtgtg tgtatatata tgtgtttgca tgtgtgtgtg tatatatata 58620 tagtatatac atattttttg agacagcatc tcactctgtc gcccaggctg aagttcggtg 58680 gctgatgaca gctcactgca cctcccggct caagtgattc ttccacctca gcttcctgaa 58740 tggctgggac tagaggcgtg tgtcaccaca cccagttagt tattttattt ttcgtagaga 58800 tggtggtctc actgtattgc ccaggctggt ctcgaactcc tgggctcaag cgcttttcca 58860 cctcgacttc ccaaattgct gggattacag gtgtgagcca ctgcaccggc ccatccttta 58920 ttttaatatt atgcagtgcc ctgagacata taaaaaaccc accttcccaa gtaaaggaaa 58980 ttcaagctga tgcctgcaga gccttcttta acaaaggctc tgaaataccc tctctcatta 59040 aaatgatact ttccaataaa attttgttta acaatgattt acaaaatgat aaaatttatt 59100 tattttgatt gtgtatggat catggtaaca ataaaaagac ttgtaaaaat aactaaattg 59160 aaagaatctt gaacatttag agccttaaga ctgtaggaat tgaagaccac agaattatta 59220 atttatatta atatttttgt tgcagagaca taatgaatga tcaacgaaag gcttttaagc 59280 gttaaaaata tattacacta gataaaatta tttgcgggaa tgggatggaa atacattttc 59340 aagagagaaa ggagcaatgt aaaatgaaga tgtaaaatcc ttctgctggt tgtccttggg 59400 gttttctttt aaagaaaagc ttggcagtgt ttttcttttt ttccattgga tgatggtgaa 59460 tatcaaatca ctttggtgct aatatttcat ttaatacatt aattttaaaa ttttctgtag 59520 aggtgggatc tcactatgtt gtccaggctg gtttcaaact cctggcctca agcaatctcc 59580 ctgcctcagc ctcccacagt gctgggatta caggtgtgag ccactgcatc cggccccatt 59640 taatacattt aaaagagtgg tgtaacaatt tttatttaaa atgtcatatt tacaatattc 59700 tagaatgtat atcttttcaa ctcattaaac ctaaacatcc ttgtaaaaag tgtgaaaagt 59760 tatatagttt ttcaaaattc gattagcagt tacataagca taaatgttta aagtatgtat 59820 ggtacagcca ggcttcagtt ccctgtctta aacacaaaga tccatatcaa ttccagatac 59880 tgcaatggtt tgctgttttt cctgcttccc ccatctccaa ataaactaaa gcatcaacat 59940 gcctcacctc acataaccct aagttttcag cagttggcag ttacacctgg aaaccatttt 60000 tctaaaataa acaacaactg tttgcttacg gatcaaaatg caaaggacca taacatttag 60060 cctcaccttc ctactacaga tcgagtttaa aagtgccatg gtatagctaa attatgaaga 60120 aagatatgaa tataactgca aaagtggaag gagatttggg ataattcttg cccattttgt 60180 taggccaaat gcatctttgt gcaaattaga aaaaggtggt cttcatccct tcactcctat 60240 ccttttgggg gtggaggggc agtggctaaa gtacagacta ggtttcagct accacatcct 60300 ccttcagtta gctgccctcg gcgtgacaga aacatgtgca aacagccctg tgcctttgtc 60360 ttatgttcca gccagccaag aaaaatagtt gtaaaagagc agctgctgtt tggggtaatg 60420 accttggacc ctccccaatt tgttccaagc ctgtttttgt attcattttt cccacattta 60480 tgttcctgga tggaagcttc catatctgct cttggcccta tttgaaattc cccagatttc 60540 cttcctggct cctggccttt ggtttttcat gtggctcctg atcccacacg ctccctgaat 60600 ttggattctc ctgtcatttc aggtgcgagg tttcccacta cagcctcttg ggcctcacct 60660 ccaatacctc tttcccatca gaacagcccg gaccttcccc tatggtagag cagagacaga 60720 atttaaatga attctcaaga agtgcttgga ctcatatcta gcaaaattac atggcattta 60780 acctttgaca caaaaaatgc agcttctagg aatctatcta aagatacact gtggcaaata 60840 tacaaaaaga agcattattt atcaagcact atttcctaat aaaataattc ttaggtcagg 60900 cgcaatggct caggcctgta atcccagcac cctgggaggc tgaggaaggc agatggcttg 60960 agctctggag ttcaggacca gcgtgggtaa catgacaaaa ccccatctct aacaaaaata 61020 caaaaattag ccgggcatgg tggcatgcac ctgtagtccc agctactcga gaggctgagg 61080 tgggaggatc gcttgagcct gggaggcaca ggttgcagtg agccaagatc gcaccactgc 61140 actccagcct gggtgacaga gtgagaccct gtcaaagaaa gagagagaga gagagaaagg 61200 aaagaaagga agaaaggaag aaagagaaag gaaagagaaa gaaagagaaa aagaaagaaa 61260 gaaggaaaga aagaaagaaa aaagaaagaa aaagaaagag aaaggagaaa aagaattctt 61320 actaataaat gcaggagaaa tgatagaatt gaaatatcac cattttcaat tcctaatgaa 61380 ataacgtatc taggcaatga ccatcaatag ctagatgcta aaatcatctg atcaaacact 61440 gatgggaact tcgtaacaga tggatcaggc taacaacatc tgaaaccact aactggtttt 61500 gatgtcataa aaagaaaaac aaccagatat tttctgtctc ctgatgagtt gcaattggag 61560 ctacatatca cctgtaaagt cttctggcca aaaaattaag cccagccgga ccttattaaa 61620 cctttaaatc taacaattag ttttgaagct tttacagatt aaatgaagtc tgagatttgc 61680 ttcaaaatga accagtggtg gggaggaagt gggtgaggtg taggtgaaac aagattggcc 61740 acgtcgataa ttgctggagc tgggcgatga aagcacaggt atttatcaca ccatctctct 61800 acttttgtgt gtttttttgt ttgtttttgg ttttggtttt aaggagcaga gagtctaata 61860 ggcaagaaag aaaagagaag gctgaaggaa gacgctcccc cgtacagaga cagagggagg 61920 gggctccaaa gccgaaagag gaggtcctct tgtgtatgtt ttaaaatact cccagataaa 61980 atatttttgg aagagtactt ggttggattc aacagctttt ttttaattta aaaaaatcac 62040 ctcaattttt ttgcttgctc taacgtgcca tagaaattcc tgaggtttta cttgttgctt 62100 tacaatgaac tgtgtaaaca caagctggaa gagatcagct atgcgctgga agggttggtt 62160 aaatattgag actgccttgc tgagggaagc cttttaatga atctcagtaa ttttgcaaga 62220 gaaaagataa caatgaacac tacattaaac atcattcttt tgcactttgc taaatatgtg 62280 tatgtaaatt actgtctgac tgttactgga tatatacagc atatacatat gcactttttt 62340 tactgttttt tttttttttt tttttttttt tttttttaca gagcttgctc tgtcacctag 62400 gctggagggc agtggcgcag tctcagctca ctgcaatctc cgcctcccag gttcaagcga 62460 ttctcctgtc tcagcctcca aagtagctgg gactacaggc gcctgccacc gctcctggct 62520 aatttttgta tttttagtag agacagggtt tcactatgtt gtccaggctg gtctcgaact 62580 cctgacctcg tgatccatct gcctcggcct cccaaagtgc tgggattaca ggcataagcc 62640 accgcgcccg gcccatgtat actatttata catttttagt atcattttgt ctttacattt 62700 tacataattt cagatacatt ttcctcatat caaataattc agcatttttt agtactaaca 62760 tcatagtctg taagccattc aaaaaatgta tttcacaaaa taggctatct catcctttga 62820 gctattgaga tgaattaatt tatactcctc ctaagatccc tctcgtcact aagattcttt 62880 tattttatga caaaaccata gttctagaag cttgtttctc ccacctgaaa agactggatt 62940 tgggacatga tcctgtagaa cttcggaggt aagcctggtg aatcagatca tagggggtct 63000 ggagggtgaa aaaaaagggt ttggtgctca tggatggggc tagtattggg gtgtagggga 63060 gattaggtca aagcaagagg attcaaagga gaaatgaatt cctttagatt ggggaagata 63120 atcggaagag gtaaaagaca ccgtccatga cacttcctgg ggaagcagat gtatgtataa 63180 ggatgtgagt attgtggttt tgtaaagaat gcattcctga agatgttgca taatttaaaa 63240 cctacatatt ttgattaatt ttctcatgag aatagcaggg tatgtgttct cggcgctcac 63300 aaatgtataa tccattgtgg caaatttttg ctttcacata ttttttttta tcattattgt 63360 cacaggttct gtgacggagt tctggtttct aaattcacag cataccaagg cagttcttta 63420 aagttcttga tactctttta tcatatctaa cttgtattcc aaaattattg agttggagca 63480 cattttccca gcacttagca ccgctatttc atggatggtt ggagaggggg tccaaaaatt 63540 ttacaattat gttaacaaaa gtaacacagc aacaaaacaa taagcaaaat cactgccaga 63600 gtattcctta gcttgaaaca atacggttca catcgataga atatggcatc tatttctgtt 63660 taatcagtta accctgctaa gtagcaagag cttacaattc atgtctaaaa tcatgatttt 63720 tttactagtt ttttaaaaaa tgtgggctct atatatataa tttaacattt tgcttgtaag 63780 acttaatttt gcctgggtat ggtggctcat gcctgtaatc ctagcacttc aggaggttga 63840 ggcaggagga ctgcttgaac ccaggagttc aagaccagcc tgggcaacac agtgggaccc 63900 catcactacc aaaaaaaaaa aaaattagtc aggcatggtg gtgtgcactt gtagtcccag 63960 ctacttggga ggctgaggtg ggaggatcac ttgagcccag gaggtcactg ctgcagtgag 64020 ccattattgt accacaacac tccagcctgg gtaatagagt gagactctgt ctcaaaaaaa 64080 aaaaaagact atttctaaat gtgtggctat attataccat ataaatgtgg cttcttgggc 64140 aaggaaagag gacaatatag atgaaaaaga aattgatcct accagaagtg atccttttat 64200 ctgcataact ctcaggcagt tgtggcaaat aattggcaat atctattgtt ctgaaactgg 64260 ttttcgcaac ttttattggg aacaccatcc cctctcctgc atgatcagtt tctcctctcc 64320 acggatcatt cacatgagta aagtcagtag cgtgctggta aatgtttaag atcttgttct 64380 ttgggggaaa aagttcctaa gttctagcag ttgccctgga taacttcaag gtatcaacat 64440 ggaagttatg tacaaaaatg gctgtcacaa gccagtatga gctaacacca acatactacc 64500 cagtgttctt caaacttcag ctcacagccc attagtgggg cttgcaaaca ttttagtgga 64560 ctataagcag cattttttta aaatgaaaaa gtagattgtt ttacacataa caggagtatt 64620 gttttgtaca attttttttt tttttttttt tttttagaca cagtttcact ctgttgccca 64680 ggctggagta cagtggtaca atctcagctc actgcaacct cggccttctg ggttcaagcg 64740 attctcatgc ctcagcctcc cgagtagctg aaattacagg catgcgccac aaggcccagc 64800 taattattgt atttttagta gaaacagggt ttcaccatat tggccaggct ggtctcaaac 64860 tcctgacctc aagtgatcca cctgcctcgg cttcccaaag tgctgggatt acaggagtga 64920 gccaacgtgc ctggtcaaaa tttttgtttt cgtaatttta agtatgtgtg tagtaagtct 64980 caatggaaat gtaattctta tggcaggtca cttgaaaaaa aagaagtcta aaagtcacca 65040 atgtagtatc ttctctttaa aaaaaaaaaa aaacaacagg agaaaacctg aatctgccct 65100 ttgctccact ccttcctcag ctataatgct gcttctccat tcctcctcac agcaaacctt 65160 tctgaaatct ttatagtcat ggtttccacc agttcttcac ctcccatttc tcaacacact 65220 tcagagtcag agtcagcaat gacatccatg ttcctaagcc cattgcttac ttccgtcctc 65280 cttggcctct cagcacactt ggcacacagg ctgtttctct ttctttggca tctgtgacac 65340 cactctcagc aaattcccct ggttgtccct tctcagtctc atttattggc gttgtcttat 65400 ctccccaggg ctgtccgagg tgattttctc ccactactct cctaggtggt gccatccaat 65460 ctcatgatgt catatcattc ttccctcatg cttcagccat actggttggt ggcctttgtt 65520 tcctgaacac atttaatgca ttctcaagac cctcagggac tttgcagcag ctgctcgcta 65580 aggctggaat gctcttcccc accatcttca tatggctgtt tcctttcttt cactcaccag 65640 cagcttaaac tttgactcct ctgagagact ttccttgtca cccaactaag gttgccactc 65700 aggtgctccc aatttaatct tctctaaaac acatcactgt atgtgtcctc aactagagta 65760 taagcttcct cgaaacaaga acaatcaaaa ctccttgccc tcatggagtt tatagtctta 65820 tgatgggtga agtaacataa aataaaaagg caccttatat agtatattag catgacaaat 65880 gttagccaga aataaagcaa ggaagagttg ctagggagtg tgtatgagtg tgttttggga 65940 gagtgtttgc aattttaaat attggtggtc aggaagggcc ccactgagaa ctgacatttg 66000 agtagacttg aaaagggaaa aggaaatatt gagtaaagat tttaggatgg gagtgtgaca 66060 ggcctgctag gagaatagca aagtcgctgt ggctgctgca gaaaaagtga gaaggaaagt 66120 agtaggagat gaaatcacag tgtgtgagga ttcgggcaga tcaggaagtg ctcgtgtaag 66180 aactggatct ttactcaaag aatgagcaaa aattagtaga cggttggccg gatgcagtgg 66240 ctcacacctg caatcccagg ttacaggagg ccgaggcggg cggatcactt gaggtcagga 66300 gttcaagact agcctgggca acatggtgaa acctcatctc tactaaaaat acaaaactta 66360 gctgggcatg gtggcgcgca cctgtagtcc cagctactca ggaggctgag gcacatgaat 66420 cacttgaaca cggtaggcag aggctgcagt gagctgagaa tgtaacactg tactccactc 66480 caacctgggc aacagagtga gactgcctca aaaaaaaaaa aaaagtaggt tttagtaagg 66540 gattaacatg atctgaatta tgttttgtca tgacttctct ggctgttgtg ttgagactac 66600 attgcagagg ggcaagggca aatataggga gaccgattag gatactgcag taataatgta 66660 agagatgtgg gactctatct agaagggccc atgaggtcct ttgcatgcta gtattcttta 66720 ctgctgtgcc tggccatgat aggcattcag tgaatatttg cttatttaaa ataacacact 66780 gggctaattg aacaacagtg ccaaatgagg gagatatttc taggaataag ttcttaggat 66840 ttatgaacat tttaatccag attttctttg ttaactctgc tctctggccc tttcactcag 66900 ccccgtttgc acctaaatat gacttacaaa agaaacacag catttatgtg tacttatttc 66960 aacttacttt agctttgtaa agaagtacaa ggttgactca gggcccagct tggtgtctca 67020 tgcctgtaat ttcagcattg tgggaggcca aggcaggaag atattgtgag cccaggagtt 67080 tgagaccagc ccgggcaaca cagtggaccc tgtctctaca aaaaaaaatt tttaaattag 67140 ctgggcatag tggtgtgcgt ctatagtccc agctactcct ggggctgagg tgggaggatc 67200 acttgaggga aaccctgtct caaagtggcg gggctggggg gagactcagg cagaattgtg 67260 aagatattca attgctcctg actttatcaa taatctaaca tttcaaccta acattgatat 67320 ctattttatg caaagcatta cactatgcac tggagactgt ataagacaag ttccttttct 67380 caaactacag tcgagttgga tagataaaac acacaacaca taccaaaaga cagctataaa 67440 tccaaggcag tgtatgtcaa gggtaaattc acctattcag attggatctt gagaagtgca 67500 tcaggcttgg aaaatgggta aggaggagag aaaagcaaca gtgaatcaga acatgagttc 67560 ccagttatgg gacttgtaat gaattcctca attaaaacaa aaaataatga aaacaaaagc 67620 cagggaggag aaagcccacg ttaatgacac taaaatatat ctttccaaac aaatgtggat 67680 aaaagccaag tagagaagat gagaactttg aggtccctaa cacaaaataa acagtaagca 67740 gccagccatt ccaagtggct gacatgactt tgtttaactt tatttgtatt tctggctggt 67800 gtgtttacag ccaataggtc aaactatcag tcagtgtagg gccctgagaa gtcgggtatt 67860 taagagcatc taataggcac agaattgtgc tccatactgc ttaaactgtt ccctaagtgt 67920 ccaatttgga gaaaacaccc acacgcagga taaccggcga gtgacgcgga gtggctgcga 67980 gtccaagtta tcactaacgg atggggagct tgggctgggc acagtccagc gtactgaacc 68040 cttcccccac cgtttcacct gcatacagag gtgtgtactg tcaaaaagca gcgcctccaa 68100 gtctcttctg gcactgtctg gacttggatc cgaggcagac gaggaagctg agaaaaccct 68160 ggcgttgacc ccgtggacct gggcgccccg ggaaggccag cgcttggtcc aggcaggcgg 68220 ggcctgtgcg gtgaccaccc tggtcctgaa aagtcccagc cccgagcgcc ctccctccta 68280 gacctggagg cctggaacag ccaggtggac gtcggcccac ctttcttttc tttccttccc 68340 attttcctac cacctcccac cccactccgc cttccgggca aaggcagcca gatccaccca 68400 ggacacattc tttgtcctta tccctctgtg ctcgtcccac agcaagccag tcgcggtcca 68460 aggctccaga ggctgtgcag gaggccgagc tgggtggcga tcagcggcgg gtccctgtcc 68520 aaaatccagc agagccgcca gggacgcccc agacacagaa ggcggggcgc ggggagggtg 68580 gggagaccac agcagtgagg cgcgcgagcc gggaagtgaa cgaggactga ctcctgtcgc 68640 ttcccgtagc cgcccacgga cgccagagcc gggaaccctg acggcactta gctgctgaca 68700 aacaacctgc tccgtggagc gcctgaaaca ccagtctttg ggtgagtcgc gcgacccccg 68760 gcctcgggtg gcggggcagt cgctagaggc gtggctgctc tgagggtctc gccagtggag 68820 gatggcattc ggatgtcacg gctcctaaat caccatttga tgggtgggac agtgtccagt 68880 ccaccccgac ccgccggtcc tcaccgcggc agagccgggg ctgggtggcg gggacgctgc 68940 ctctgcaggc gaggcgctcc ggggcataag ggattatcag gagtcgcggc ctttcttgga 69000 catccctggc tggggtcagg ctgtttgccc tggggtgtct cctcgctgca aacccacccc 69060 acctgggctg ctttctcacc tgttccctcc tagcctgagg ccgagcgcca cctccaagtg 69120 gaggaatctg gggaagtttc cttcccggaa tttgtagtga cagtggagtg acctccattg 69180 cgttccctgc ctctaacacg ctctttagga tgccgagtca tttgactgca gtgttaaaca 69240 ttgcaaagcg caagtcatgt gacttccttt gaccgtacgt gaaacttaag tgatggctgc 69300 ttgtgatgca tacgaagtgt tcatgctggc gggacctgtc cctggggata cttcgggggt 69360 tgcgtgattt aatgcaagca gatggcttaa attgggtcac tggcttgtta ttatacatgt 69420 gtatggcaac tcggcatcca ttctttttgc tcttgttctt acttcctgaa ttgagtcacg 69480 gagccagagt tttgaggttt tgactaacga attaagttaa tgacatgggg ctatatttag 69540 gtggtaaacc aagagggata cagttttttt tcttaataaa gaaaaagtga tagatttgat 69600 cggtgtgtat tgttggtgtg cagtataatg acagaattgc tggaagtaaa atacaggaag 69660 ctctggtttc atttcccctt tagttctgct taaagtcgag tttttcctgg agctattaaa 69720 tgtagtgtag tgtccatgag tgcttttatc ttaaaaaatg tggctgatgc tttccaacac 69780 tcccctgccc tgtgattatt atttttttaa gcaacagaga aaactgtatc ttaatagtat 69840 taaaagtatt ggatttttcc ctactttgat ttgtttaaat tggaggagga agagcaattc 69900 tttctattca caataataat agctaacata gcgcttactc tttcgctgtt ttattaactc 69960 aatcctcaga acaaaccaat gatgtgaata ctgtaattct cattttatgg aaatgaaaat 70020 ttaaatgaat acctctgata attgtacggg actgtttgat tagtatttac cattaattaa 70080 ttaaattttt tttttttttg agatggagtc tcgttctgtt gcccaggctg gagtgcagtg 70140 gcacaatctc ggctcacttc aacctccgtc tcccaggttc aagcaattgt cctgtctcag 70200 cctcctgagt agctgggact acaggtgcat gccactacgt ctggctaatt tttgtatttt 70260 tagtagggat gggatttcac catattggtc aggctggtct ccaactcctg aacctcaggt 70320 gatccacccg ccttggcctc ccaaagtgct gggattacag gtgtgagcca ccatgcctgg 70380 cctattttag tatttttaat aataaattcc atgttagaaa ttttctactg atgtattttt 70440 taagtcaata tttcctacac tcacaatcca aaattattta gtatatgagc acactggtaa 70500 gaatgggagg cagatcgttg attgtaataa tattctatta tttggtaaat atcagtaaca 70560 taatatataa tttaaatttt aaaataggat atgaagaaaa atgctacatg cttacttttc 70620 ttttcctcta tttttacttt acacagggcc agtgcctcag tttcaatcca ggtaaccttt 70680 aaatgaaact tgcctaaaat cttaggtcat acacagaaga gactccaatc gacaagaagc 70740 tggaaaagaa tgatgttgtc cttaaacaac ctacagaata tcatctataa cccggtaact 70800 gatttctata agataacttt ttacctatgc caggacagat ccaatagaat attaattatc 70860 cattgggaga cagggcaaga ataaaagcca gtgaacatat ttaaagcacc tactatgtaa 70920 tagagatggt ggtgggtgct gattacgaaa cagctcttgt cctctagtgg aggaagaagt 70980 cacaatgata atatgacgtg atgaaacagt gttatgaaca gggaacgtct gggtagagtg 71040 gagggaatgc caacttttgg tgatgggagg aggctcagct aatcataaat tgtagttttt 71100 aaaggaaaat ggatttctta ctctacaagt ttttcatttt cttttttaat tagagctgtc 71160 catgagaagt taatgtctcg atctttccct cagcctttca aatactgctt ggcccttgag 71220 cagggaaaat gtcaaaagcc aatggggaga tggagagtgt gaagtagtaa gggtctcgtg 71280 cagttcaggc aggtcctaga atccctgaat gactgtaatt gctggaaatt gccctgtaat 71340 cctgagcagt aaagagcttg ttttagtttt atgtggtggt gagaatcttt aggaatgtct 71400 agtttccacg tatctgaagc tgaatcctga atcgaggtct gaaaaaggac agccactttt 71460 ttagtaaacc gcctagaaga ttcttgggca aaaggaaggg tgagaatcct taaaatgagg 71520 ccctaaacca gttttgttag tgtgtgtggg ttcaagtttt tgtcatttac tttatagctg 71580 tatttccttt ttccctaagt tttaatgtca ttgtgtaaga atgaggtatc gctgctgtat 71640 caagcaaagt cagttttagg agaaatagcc tttcagtggt agtaagttta aaaaagatga 71700 cttcctgaag cggaagcttg tgagacattt aagatgactt tgcgcatgtt agagttaaaa 71760 acatcccaag gttgtaaact gatttcctgc aaagatctta acaacaacaa caacaacaac 71820 aaactaggct gcctgccacg ggtgtctgaa gtatcatctt ggctcaagct gggagaatgg 71880 ataaaggtta cactgttcat ttctgccctt cacacagaaa agaagataat tttataggta 71940 aaattcgtgc atatcttgat tctagcatac tgctgattcc tgtagtttct ggggtcagta 72000 ctctcaacta ttgaggtgga acaaaaataa gtagacttca tttcttgagg aaggggatct 72060 ggagaagtag ttctgcgcta gagcagaaaa tgccttcagt cttgtggcat gggctggatg 72120 ctgttctgag gataatgcat ttccaaggga gatatttttg gcaaatagct tttttttctt 72180 tcttttcaaa attctctgtt ttattatcag ttctcacaaa agagtcggaa aggttagagg 72240 tagactgaac tgaatggcaa aaacattttg cgctctcttt acgtttcact gctgtaaaat 72300 atttatagta taaagggcct gtattgcact gaatttctct catttgtagc tagttgccct 72360 ttcaatgttc caaaaaaaag gctgtaaata acttatttta tttattcaat taattttttt 72420 ttttaaattt tttgagatat agtttccctc tggtcaccca ggctggagtg aaatgatgca 72480 atctcggctt actgcaattt ctgcctcccg ggttcaagca attctagtgc ctcagcctcc 72540 tgagtagctg ggactgcagg cacgtgctac catgcccggc taatttttgt gtttttagta 72600 gatatggggt ttcacagtgt tggccagcct agtctcaaac tcctgacctc aggtgatgtg 72660 cccaccttga cctccaaaag tgctgggatt acaggcgtga ggcaccatgc ctggccaact 72720 tagttattta aagataatca attagtatat tttataagct agacttagga aaactgtttt 72780 cagctgggca tggtggctca cacctgtaat cccagcactt tgggaggccg aggcaggtgg 72840 atcacgaggt caggagttca agaccagcct ggccaagatg gcgaaactcc gtctctactt 72900 aaaaatacaa aacttagcca ggcgtgatgg cagcctcctg taatcccagc tactcgggag 72960 gctgaggcaa gagaatcact tgaacctggg aggcggaggt tgcagtgagc cgagatggtg 73020 ccactgcact ccagcctggg tgacagagcg agactccatc tcaaaaaaaa aaaaaccccc 73080 cccacacaca aaacctgttt tcttgaatca tggttgtttt gttactgata ggttcaataa 73140 gtaaatatat ttattgtctg ttgtattctt tattaggcat tataaacaca ccgccacttt 73200 ttaattttta tttcattaat gtttccaatt tttttttttt tttttttttt tttaagacag 73260 aggctcgctc tgtcatccag gctggagtgg agtggtgcag tcttacccca ctgcaacctc 73320 cacctcctgg gctcagcctt gtaaatagct gggactacag gcatgcacca ccatgcctgg 73380 ctaatttttg tatttttttt ggtaaagaca gagttttgcc atgtttctca gtctggtcaa 73440 gcactcctcc cgcctcggcc tcccaaagtg ttgggattac aggcatgagc caccatgcct 73500 ggcctatttc taatattttg gtccacattg gtgttagacc aactgtccac attaagtttt 73560 cttggaaaag atgaagtaaa tattgcaact ggcctatgta tttttttccc tatttagtat 73620 atttctttga ctagttcaac tgatagaatt ccaagactta aaaaagtcag gctctaaggc 73680 tgggtccaga ggctcatgcc tgtaattcca gcactgtggg aggccaaggc tagtggatca 73740 cttgagccca ggagttcaag accagcttga gcaacatagt gagaccttgt ctctctataa 73800 aaatacaaaa attaactggg gattgtggcg catgtctgta gtcccagcta tgaggaagag 73860 tgaggtggga ggattgcttg agcccaggag gttgaggctg cagtgagctg tgagtttgac 73920 actgtgcttc attctgggtg acagagcaag aaccatgttc aaaaataaaa ataaaaagtc 73980 agagtccggg tgctgcggct catgcctgta atcccagcac tttgggaagc cgaggcgaga 74040 ggatcacttg aggtcaggag ttcgagacca gcctgactaa cacagtgaaa ccccgtctct 74100 actaaaaata caaaaattag ccgggcatgg tggcggtggc ctgtaatccc agctacatgg 74160 gaggttgagg caggagaatc acttgaaccc gggaggtgga ggttgtaatg agccaagatt 74220 gcacaactgc actgcatcct gggcgacaga gtgatacttc atctcaaaaa aaaaaaaaga 74280 aaaaaaaagt taggcttcct tttctgtttt ttttttcttt tttcttctct tttttttttt 74340 ttttttaaga gatggaggct tgctctattg cccatgctgg agtgcagtgg tgcaatctcg 74400 gctcactgcc acctttgcct cctgggttct agcaattctc ctgcctcagc ctcccgagta 74460 gctgggacta caggcgcaca ccgccacgcc ccgctaattg ttcttttgta ttttagtaga 74520 gacggggttt caccatgttg gccagcctgg tctcgaactc ctgagctcag gcaatccgcc 74580 cgcctcggcc tcccaaagtg ctaggattat aggcgtgaac caccgtggct ggccacttac 74640 ttttctttct attgaatttg aatgaataat ttggaagaca gtatctttac ttcataccag 74700 gaatgctgcc agtgaaattt cttgtttggc agttcattat ctacctatat atttaatttt 74760 gctattgttt atagagttct taagatatga ttaaatgcta gctggttaag aaatcattta 74820 gaaatgaaac agaattggtt gttactccaa gttaataagt tgcttgtcaa cataaatcct 74880 acctggtacc cagttttctt aggaaccttg cttccatgtt tatccttttc tgcttagtat 74940 tctaagtact ccttttttac cttacaattt agtcttaaaa cacaacacag tcaagtcttt 75000 cttttgtaac ctgtgaggta ccttctagcc tttgtgctgt ttttcttctt tttttgctgc 75060 ctgccttcct gactgagagt ggatttcctc actaaggctc tgccctctga tttttcactc 75120 tcttttcttt tttggtttta ctagtgaaat tttgtcttta atgtctcttt cttttatgtc 75180 tttaccgatc actcataaat ttttttttcc atatgtatcc agttccaacc tttcacctaa 75240 tgtgaacccc caactctcag ttgctcagcc agcccttcaa gactaggagt tcaaaaccaa 75300 acttgcatct tccttcccaa accagctttc ctcttgcagt tttctgcagc aggatccttc 75360 tgctgtttaa cttttgcctc ctcccttgtt tcctagcacc caatagttgg aagatagtct 75420 gtcttcaaaa ttttaaacta catttatgtc caaaccagtg gcttttcctt ttaaaaaaat 75480 ttaaagataa tatgtgcaaa tcattttttt aaaattcaaa cagtatttaa gagtttcagt 75540 gaaacatgca ttttccttct accctggtac ttagttttac tccccaaggg caatcacttt 75600 ttactggttt ttagaaatat atccttcctg agatacttat gaatatccaa aagtgtgtgt 75660 gttgtgtata tcacctttta tatatcctgt ctctttacgt gcatgcattt taccgtataa 75720 actgttttct accctgcttt ttctatttga cctattttgg aaatgtcatt ttatttagaa 75780 cttcctcatt tattttaaca gctgcataat tagcagtaaa acttatgtaa gcagtccctt 75840 gtgaagggct gtgtcttttt gcgattatat ccggtgctat agtgtacatc cttgtgtgtg 75900 catcttggtg tgcctgtgct acgtatttct gtaggataaa tctgtaaaag tggaatcact 75960 aggtcagagg gtatggtcca ttttctttac ttatttattt tatttattta ttcatttatt 76020 tttgagacag agtcttgctc tgtcgcccag gctggagtgc agtggcatga tcttggctta 76080 ctgcaagctc cgcctcccgg gttcacacca ttctcctgcc tcagcctccg gagtagctgg 76140 gactacaggc gcccgccacc cacgcctggc taattttttt gtatttttag tagagacggg 76200 gtttcaccat gttagccagg atggtctcga tctcctgacc tcgtgatcca cccatctcag 76260 cctcccaaag tgctgggatt acaggtgtga gccaccgcgc ccggccccat tttattatct 76320 ttatttgctt ggatccttct tagcttcttc aatgttaaag atattgacag ttttcctctt 76380 actgaaattt ataaatccat tgactccctt gatattattg ccctggcctg actgattctt 76440 ctctctcctt tctcttctca ccccatgttg aggtccccaa ggtcacaccc agttttgatg 76500 actcaccagc atagagttgt acttgtgcct atgatttatt gcggtgaaag gatatagagc 76560 aaaattgcaa acggaaaggc acctggggtg aattccaggg gaaatccagt gcaagttcca 76620 aggtcgcctc ccagtggagt cacataggat gtgcttacat cctccagcaa ggagttgtga 76680 caacacttgt gaaatgtgga ctgccaggga agctcatcag agcctcagtg cctagggttt 76740 ttactggagg ctggtcacat aagcaccctc acacatatca aaaaattctg gtcccccaga 76800 aggaaagcag gtgtttagca taaccatatt atttgcatga acagttcagg tacaggaaat 76860 ccccgttacc agttaggttg gtgggtgccc ttctcaaatc ccaagttccc agacaccagc 76920 caggggcctg cctcgtaagg aggcctttcc aggacagcag tcaggcctgc caatgttaat 76980 tcttttctgc atacctccta attttagaaa ccaccgagcc tttgctgcct gacctgtcct 77040 gcttttcgat ttctttatct actttgatat ctttacaaat gatctttacc ctgactttta 77100 aatgtgtgct ctggccattc acctagcgtg tggttctgag tctccaagtc ttagcagatt 77160 tgctctcaga tgctctgcca acgcttcaca ccaagtatta caaactaaac tcgtcatctt 77220 cctcctgaaa cctgtctccc aggccaggcg cggtggctca cacttgtaat cccagtactt 77280 tgggaggccg aggtgggtgg atcacctgag gtcaggagtt cgagaccagc ctggccaact 77340 tggtaaaacc ccatctctac gaaaaataca aaaaaattag ccaggcgtgg tggcaggcac 77400 ctgtaatccc agctattcag gaggctgagg caggagaatc gcttgaaccc gggaggcgga 77460 gattgcagta agccgagatc acgccattgc actccagcct gggcaacaaa agtgaaactc 77520 catctcaaga aaaacaaaaa acaaaaaaca aaaaacctgt tttctcccca gctttgtcat 77580 gtatttagtg gccttatgta gacagtttcc tttgaaacat ctcttggact tctctgctct 77640 tccagggcca ttgccaccga cctggaatgt gtccttatcg tttcacgcca ggcttatggc 77700 agcagtcagt cacccagatg acctcctgac ctctggctta tttcaccccc actggactgt 77760 tgttcctaaa cacttctttc gtatgtcact ctaaaatctg accctggctg tacctttctt 77820 taactactcc ctgactgcgt gctgagagaa gatgggtctt gtcttttcct gcctctctgc 77880 ttttgtaaac tgccatttct acctgaagtg gcaactgaaa tcatatcttc ttcataaact 77940 gtctttggct acctcagtta gaattcctta tcccattttc ctgaagcatt tctttgactc 78000 ttctttactg ctcccccacc cttttttttt tctttgagac tgaattttgc ttgttgccca 78060 ggctggagtg caatggcccg atctcggctc attgcaacct ccgcctcctg ggttcaagtg 78120 attctcctgc ctcagcctcc tgagtagctg ggattacagt catgtgccac catgcccggc 78180 taattttgta tttttagtag agatggggtt tctccacgtt ggtcaggctg gtcttcaact 78240 cccaacctca agtgatctgc ccaccttcgc ctcccaaaat gctgggatta caggtgttag 78300 ccactgcgcc tgacccccat tttttttttt tttaaagatg ttgaattggt cagggtttgt 78360 agttacaagc aacagaagcc aactctttaa gcagaaaagg aatttgctaa atgatagtgc 78420 agagttctca gaatctctag caggatgaag aaccaggctt ggagaatagg tagccacaga 78480 tacacaagca tactgtagga cggttcccat gaagaggcat ctgttgtcac cactggacac 78540 agatggtact gtgtctctgc tactctacca atgccactgc tgtctctgac cccagatgta 78600 gctccctctg accctggatg cagctccctc tgaccctgga tgcagctccc tctgaccctc 78660 gatatagctg cccctgaccc cggatgtagc tgcctttgac cccagatgta gctttctcca 78720 aacccagata tagcggctgc ccccttgcca gagtgaatac tgcgtcattg tggcttcttc 78780 ttgtcactgg ttcttactta aaagctgagc tggaagttct aatgggcagt tttgtcacct 78840 gctcttacct tgttgcagtc tagatgaggt ctaatgttca taagctaggg gattttcaga 78900 tatggaaagg gataccaatt ttcagcagcc aaatagagta tcacattttc actccatgtt 78960 tcctgggtgt ctgttatgtt tcctgggtgt ctgactctta ggcttctttc aagctgcagt 79020 ctgcctaata gagagccttg catttaatca tcaaaaaggc aaagcaatat gaatcagcaa 79080 gggtgttttg gcaaataaca gcaaacctga ctgtggcgta agcttgtggt attgtctcca 79140 gtgtgatcag atctgtattt taatttttta aatgtaaatt aataatgatc tgtgaatcac 79200 caaagtagct tggagtagcc tagaaaacaa tgtatgtcct ccgttttcac agaagccaca 79260 tagtcgtggg ttaaatgagt cagcggcagg gcactgtgtc tcatagttaa aaaaaaaaaa 79320 aagtattact gaagtaatgc aggatctttt ctgaagtaga aggcatgatg aacccagaaa 79380 actaaagcag caagtggcca ccgttcttag catagttgtt tctcaaactg gaacaaccta 79440 taaacagttg tgaacaaggt attagaagtg atgggggccg ggtgcggtag cttctcccaa 79500 agctcattac ctcccaaagc aaccccagta ctttgggagg atcactttga gcccaggagt 79560 tccagaccag cccggccaac atggcaaaac cccatctctc taaaactaca aaaaattagc 79620 tgggcatggt ggcacatgcc tgtagtccca gctacttgga tggctgaggc aggagaatcg 79680 cttgaaccgg gaggcagagg ttgcagtgag ctgagatcac gccactgcac tctagcctgg 79740 gggacagagt gagtctctgt ctcaaaaaaa aaaaaaaaaa aaaaaagtga tgggaataga 79800 ttgttttgtc tcaaaaagct ctttccaaca ctaaaatgaa acatataatt aaaaatattt 79860 ttctggctat aaaaatatcg atgcttatta tagacatctg caaagtatga aaatatatga 79920 agaaaaaaat taaaatgcca tcatccccca tgaaaactat tgttatcatt tttgtctgat 79980 ttctttagtg tttctctttt tcttttttta atttttaatt tttttgagta tgtagtatgt 80040 atatctattt atggggtata tggcatattt tgatacagga tacagtgtgt attagcaagg 80100 ttttcttttt aatgtttata tttatttagt tgagatcata ctatatatgg ctctatagat 80160 tactttctct tatattacta acatttgtgt tattaaatat tctgcataaa gataatttta 80220 agatgaaatt tgatgttata aaaacttctc attttattaa gagattaacg ctatgaaacc 80280 tgctgctata tattcttgga accagctgtg acccaaaaga tcaatgtagg gatgtaggtc 80340 cttccccatt ctctacacac aaaatcagat actctgatgt gcagctgtag ccccagtcta 80400 cactgtctgt tgtatttttt gttttctggt gtcacgtgcc tcccaccctg ctcctagcaa 80460 ttgccatgac aacaaataga taattggctt ccgtaatttc tcatcttatt gcctaaggca 80520 acagagagct tgtgggctca gcttgcggtt cagcagctgc tttgttgcct ctcctctgta 80580 tgtgtgaggc ctgccagagc ccactttcca gacaggtgag agttcattca ttcaccatgc 80640 agttaccgat cgtctcttga cctgtgtcct ggggaggtaa aggtgacgag ccagttctgc 80700 cccatgcagc tcacagtcta ggcaaagcta catgcaaaca aacagaatcc aaagtgctat 80760 catgaaccct ctgagagggg ctgactcagc agcccaggga gcttgaagaa ggctccacag 80820 aggaggctgt gcctcaaggc gatttcggtt taggagccac caatttataa ccacttttct 80880 gtggcccgtc ttattttatt tcttatttct tgacaatcag aagtaccttg ggtaggtttt 80940 accatgcaca tcgtaatttg agtgagctta gtgtgaggct taacggtgtg tgggctgtac 81000 atcctggtca gatgctccag atggaggcag atggttgtga tgcaggagag gcagccacat 81060 agcacaggtc cccagccagt ggactgggaa gacagtgtag tcatctctgg ggaaggggaa 81120 tgacaagatc tggcagtgtg gcaggtccca gaaaaaaagg gctgggttct gggcagtgag 81180 ggtgcaggtt gagacctgaa tactgggtgg agccagctgt cagagtccac gcctgcagac 81240 tggactggtc cacggcaggt ggatgccatg tcttgaagac ccacaggcac ccactcatcc 81300 tcatgatcat gcagttctct ggtttctaac agtgcagtct gggttgcagt ctgggagtcc 81360 agcagagaag agcaggccct ggaatcccag gtgtgggggc gtggcttaac gtggagtttc 81420 cttcagaggc agtgagtgct tgtcattgtc tccgtcagca ttggctttgg gcctagtgtg 81480 gcctcgaacc ttctgttggg atcagcagtg gaacagtagg aaaaggaatg agtagacatg 81540 gcattgcaac aagtcttttt ttttttttct gttagaatta tcatattaag cagaagtttt 81600 gcttcacaaa ctctcagcca aatacaaaat actatgaata gtatttacct tgtgtctctt 81660 tccaaagaac tcatagtggt ttgcagctat tgcagatatc ctggccatgc ggtatgcggt 81720 tccttttttt tgtttttttt ttttttttga gacggagcct tgctctgtcg cccaggctgg 81780 agcatagtgg cgcgatctcg gctcactgca agctccgcct cccgggttcg tgccattctc 81840 ctgcctcagc ctcccgagta gctgggacta caggcgcccg ccaccacacc cggctaattt 81900 ttgtattttt agtagagacg gggtttcacc gtgttagcca ggatggtctc gatctcctga 81960 cctcgtgatc ctcctgcctc ggcctcccaa agtgctggga ttacaggcgt gagccaccgc 82020 gcccggccgc agttcctttt tatagctgtt tgaataggaa agatgacttg gaaaatgctg 82080 gattctgaga tttatgtgca gccttaaaaa gtgtagtttt tctctatcaa taatgagtgt 82140 gggttgtaat tgcttagtaa gtaattttgt ttatgtaaac gtacatttgt taaatttttt 82200 ttcttaggta atcccgtatg ttggcaccat tcccgatcag ctggatcctg gaactttgat 82260 tgtgatatgt gggcatgttc ctagtgacgc agacaggtaa aatcactgtg ctaaaggaag 82320 gagcatgaat aggctgtctt tttgtgattg tggaatgata acagagtaag gcgggagaga 82380 ccatttgata ctctgaggcc caattagctt tcatcagcag ccctggccaa ggtgctgagg 82440 agattggaat gaatgactaa ataaaggtta ttgggattta tttcattgct gtaagtctga 82500 tttcagtata aaaaaattag aactatcagc tggatgtggt gacttacaca tacttttcca 82560 gcactttggg aggccaaggc gggaggattg tttgaggcca ggagttcgag accagcctgg 82620 gcaacatagt gagacccccc ccatctgtta aaaaaaaaaa aaaaattaaa aattaactgg 82680 gcttggtggt gtgcgcctgt agttgtagct actcaggggg ctgaggtagg aggatccctt 82740 gagcccagga gtttgaggtt gcagtgagct gtgatggagc cactgcacta tagcctgggt 82800 gacaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaagaaaga aaaagaatta ggtatgtcat 82860 taaagaaagg aattgtggtc agatgacagg gagagtctag ttttagtctg acattcccac 82920 agcatcacag atctagttca gatggtttta ctgaatactt gctttggata caagctgtgg 82980 tatcattagt gttgggctca gctctgtgta cctaacacct gaagagcagt ggtttaagat 83040 gtgaaaatta agtctcaagg agacagccca ggccttttca gttaactcct tcaagtcgtt 83100 agagaagtag actccttcca gcttaccact ctgctatctt gagggtgagg tgaggtcccc 83160 tttcccatta tccttggcag ctagatttcc agccctcact tctgtgcttt gggtagctgg 83220 atgggtgcat gtggtgtttg cggggaaaca gagctggaca aaaggcaagt gcttgctgac 83280 ttttaaggca gtttctagta gccttccctg agcacttcac ttccatctta tcagcagagc 83340 tttagctgca caggcaggcc tagctgcgag ggaggctggg aaaggtaggt ttttattctg 83400 ggcagattca gacccagttc aaactcaggg gctattttac tgaggaagac agaaaagatt 83460 agacagtcag ctctttaggc ctcatagtga atgaatgagg agggattggt cagtcccttg 83520 tcactgggcc tggagtgtag tgcctgctgg gcctttactg gtggctttcc tttctgagca 83580 ctcatgaggc ccctgtgtct tccctcatat agattccagg tggatctgca gaatggcagc 83640 agtgtgaaac ctcgagccga tgtggccttt catttcaatc ctcgtttcaa aagggccggc 83700 tgcattgttt gcaatacttt gataaatgaa aaatggggac gggaagagat cacctatgac 83760 acgcctttca aaagagaaaa gtcttttgag atcgtgatta tggtgctaaa ggacaaattc 83820 caggtaggtt ttggagaggg acaggttgag tcctcattag tgagcaggag tgcacagggg 83880 ggcctttcac atttgtgagc ccagccttgt atttcctaca cctgagatat agtttggctt 83940 tgtagtcttt ctccataaaa ggaccaggaa ggcacctaaa tatgaggggg tggcaccact 84000 actctccagc cagttgttgc catgcagaaa tatggtccac tgtgactaga tctttttatt 84060 agatcctatt tctcctagca gggctgagtt ctgaattgac acagtattat gttcatgatg 84120 ggagggtaag ttataatata accgtcacca cctgaagaac taacaagggc aatcccagca 84180 tagaaatcag aagggttttg taaattcaag tcttgccaca agacagttct gtaggatcat 84240 gagattttta gacccagagg acatcctaga aatccttgat gtcagttcca tctctggctt 84300 catggagtgt cttataccta gcgcgcgtgt gtatggttga atttggtccc agaagctctt 84360 acacctgctg gccctctggc ctgtggagct ttcccacagt agaggtttgt accaacgtga 84420 gagaagactc acatgcctct ggcacagatc ctttctgatc ttcgggatac tgctcctgcc 84480 cgaaagtctt tctgaatctc ccaaactcca ttcacctctc ccttctctgg ccttttgagc 84540 ccgtgtctgt atcattcttt ttcacagttt ttaacagttg tgctttggct ttatgtgttt 84600 attttgcctc cacaatggga tttaaagctc cttgagtcag agactatatt gtatgctgct 84660 cgcgttttct gcctataacc taacgtggta cctggcattt gagagggagg gagggaggag 84720 gctcgtagcg tgccgaggac ctgcagaagc tactttctcg tcatcttact gtagtctgtt 84780 gaggtagaga ttgttcctac ttcagaataa gaaaaccgaa ttcaaatatg ttgggtaact 84840 tgtccatatt aatttattta gcaaatacaa cagattttga gtgtctgcca catgggtggt 84900 ctccagggac agtgttgtgg ggagctcgca ggcagatctt taacctgggt tcacaatctc 84960 cagggcacct gtgcctgggc ttccaggcga ccttcgaacc cagatgtctc acatgtatgc 85020 agaggcgcac acaagcacac gcacatatac ttatgactgc ctgtttgtct ggggagagac 85080 agttcctggt gcttaatcaa atcaggaact caaaagaagt tcggaagcac tgctggtgtt 85140 ttgggtgctt tcggttacca tttggtcacg tgtgtggaga cctgtgggaa caggtataaa 85200 attggacgca aggaaacatt taaatttgga taataagtta atttattaac tgtttttttt 85260 tggtggcggg ggggctctgt cttctgtatc tctctaggtg gctgtaaatg gaaaacatac 85320 tctgctctat ggccacagga tcggcccaga gaaaatagac actctgggca tttatggcaa 85380 agtgaatatt cactcaattg gttttagctt cagctcggtg agtgaccttc cacagcttgg 85440 ggtcttttat gaggatggtt tctgatgaga tggtagaaaa aatcttcaaa taacacttct 85500 attgacataa aaaggacgta tctccctgac tgtagtatta attttttgga agtgaactgt 85560 tcacactagc agaaggctgt ttatcagcca gggcttcatt gtctgtagga tctcaaacct 85620 agtgtggttt taataaaaca cacacagttt ttagctgggt agcagctatt tcctttgcat 85680 gggcataaaa tggagtattt ctgtaagaca ggttcctagg ctgggagtgt ctgagtcaaa 85740 gagcacagtc atgtgttgca taaggacagt tcagtcaaag atgaaccgca tatacaaccg 85800 tggtcccata agattgtcat atactgtatt tttaccatac cttttctatg tttaggtaag 85860 tttatatgca caaatactta ccatcctgct ctggttgcct acagtatttg gtacagtgcc 85920 tgctgtacag attcactggc caggagctat aggccacacc ctacagccta ggtgtgtagt 85980 tggcagtacc atctaaggtt gttaagtaat attctgtgat gtttgcacga tgacaaaagt 86040 catgtaagga cacatttctc agaacatacc cccttcgtta agcaacacat gactgtcttt 86100 gcattgaaaa ttttgataga tactaactcg cccttcacaa gggtaaaaac agtttgcact 86160 ctcaacagcc atgctcccac cttcttgttg acattacatc ttattctctg taatgtttgc 86220 caatctgatg gggggcggaa aggaccccag tgtcaagtat atttttggat catagttttc 86280 aagcatattt ttagtaccat ttataatttt tttatatgtc aaacaggtta tatatagaaa 86340 atattttcta ctggatgtta caaattaatc tttattatct tttctcagga cttacaaagt 86400 acccaagcat ctagtctgga actgacagag ataagtagag aaaatgtaaa tattaaatct 86460 tttaatgagc cactggttta aaaatgttgt tttagctgcc atgttaatga aatggcaaga 86520 aggctgggtt tttgaaaatt atgcttttag aacgcaagta atcacttgaa aattgagata 86580 catacttgtg gtgccaggca cgcagtaagt ttttgctgat gattcacctg tcagtttctg 86640 taactgccac tcactgttct tatgtaaaaa gcactctctc actcttaact gctgaatagt 86700 actgttctgg ggtatttcca aatattgaac atcagccagt gcactggcaa atgaacttcc 86760 atgtgtatct tcaacccctg ggagaataac tgcaatttaa aaatgcgctg ttattaatgg 86820 agaaagtgag gtcttaccga ctggcacgtt cacacctcac agacagaata gaatcttagc 86880 attctggggg caccctggaa aggacaacta agacacgttt gaagttcatg tagtgctggg 86940 tgaaggtggt ggctcaggcc tgtagtccca gcgctttggc tgaggtgggg ggattgcttg 87000 agcctaggag tttgagatca gcctgggcca catagggaga accccatctc tacaaaaaat 87060 taaaaaatta tctgggcatg gtggcgcatg gctgtgatcc cagctttggg tggctgaagt 87120 aggcggatga cttgagccca ggaggttgag gctgcagtga gccatgattg agccactgca 87180 tcccagtgtg gatgacagag taagaccctg tctcttaaaa aaatttcata tagttctatg 87240 aaaaattatt aatttatggt ggaggataaa ggactcagat gaacagggat atcagactct 87300 cttctcaacc cgtgtagccc ttcacaacac cataccattc cgtcataaag caccagctgc 87360 ctggaggtca caccagagtg gagcaggaac atcccaggct ccggccaggc tcagctcagc 87420 acaaccaaga cttcagatta taaactataa ttcttcccct tctaacattg ttgtgttttg 87480 tttcttttcc aataggttcc aaagtctggc acgccccagc ttgtgagtat ttttgcctgg 87540 gttatttcat gtggaatatt ttataaagtt gcatagaaaa tgaacagttt aaaccgtgga 87600 gggcagcttc attcattcca ttccttactg tagaactgtt tccctacagc ctagtaatag 87660 aggaggagac atttctaaaa tcgcacccag aactgtctac accaagagca aagattcgac 87720 tgtcaatcac actttgactt gcaccaaaat accacctatg aactatgtgt caaaggtttg 87780 aagagcccca aattttctta actctgtata aaaattaagt tgtaatgagc tgttacgagt 87840 aacctgtatc cacaatagaa gcccaaagca gccccctctg catttgtgtg ccgtccctgg 87900 atggattcga gagtcaacca ggcctgcctc tgagccattc ctgtgtattt cctcagcacc 87960 tccctgcttg gctgcttccc cttcaggcag aacacagtac tgcctcagac cccaggcaca 88020 gggggccttc ctggcgtgtt tcactcatac agagggcatc gggtcccacc ctgtcactca 88080 tttcatcgtc taaaatgtaa tcatgagtgt ttgcttcgag ccagggacag tgctgctgca 88140 ggggacccag ctgggaccaa ggcagactgt ctctcccctc ctgggattta cagggtcatg 88200 gctctgaaac attctgtagt gttctttgga cacgagtttt ccctggagat cgctttctgc 88260 aggcctcttg gtcctgactg tggcttcttt tcagagcctg ccattcgctg caaggttgaa 88320 cacccccatg ggccctggac gaactgtcgt cgttaaagga gaagtgaatg caaatgccaa 88380 aaggtcagta tccttcggta ccagtcacag tgcagatact tccgtgcctg ttaccgcctt 88440 ctacccgtga acggtcctgt gagctggaag tagggctagt gtcagaatct tcatttccaa 88500 agtgagatga ttcaagcagg aggtggttag attgtgaaca gccagtgggc agcagagccg 88560 actaaggccg tgttctgacc tcggcttttt ctggccagac aagagagtag catttttgtc 88620 cacgaggcct atccttgcct tgtagaactc cagagcagcc ccgtaagatc aggcaacatc 88680 ttttcttttt ttttttgaga tggagcctca ctgtgtcacc caggctggag tgcagtgtca 88740 caatcccagc acaccacaac ctccgcttcc tgggtgttca agccattctc ctacctcagc 88800 ctctggagta gctgggatta caggcgcacc accacgccca gctacttttt gaatttttgt 88860 atttttagta gagacagggg ttcaccatgt tggccaggct ggtctggaac tcctgacctc 88920 aagtgatccg cccacttcgc cctcccagtg ctgggattac aggtgtgagt cgccacgccc 88980 agcccaggca acatttttta gggcccctct tgtcatgtga tttagaaaat ttctgcttta 89040 acaacttttt ccacagacgt ccagccttct gaaagcttga aattagagct atttcctaga 89100 aagtggcata ctttcaagaa ggaaggaaca cgggtagatg atgaaaagag aatacctgct 89160 tgagaggatc ccaggctcct gcagcctgaa gtagtcattc agtttagcgt taaaccttcc 89220 atttctgtcc aaccacatct cagcctcaat gctgatttta aaggggtttt ttttttcgta 89280 tttttatttt gcaagtaacg aattagtgga atgctgactg ggtttaaaat ttcaacttca 89340 cctgcattcc catgtccatg tggatacgtg tgtttcatag agttagaatc atagttcaag 89400 tctggtcact aacattgctg aaattgccac tactctgtcc tacttggtta attaaggttt 89460 tttttttctt tctttctcaa aagctttaat gttgacctac tagcaggaaa atcaaaggat 89520 attgctctac acttgaaccc acgcctgaat attaaagcat ttgtaagaaa ttcttttctt 89580 caggagtcct ggggagaaga agagagaaat attacctctt tcccatttag tcctgggatg 89640 tactttgagg tgaggttcca gtttttgaaa atgggacagc aataagaatc ctgggagcag 89700 gggtgggata agtggtccat ttaaatcaag tcctaactca gtatgtggag gttgtgtatg 89760 ttttttgttt acttggagat tgtaatttgc cccttccttt ttataacgtg ggcaatcagt 89820 ataaatggca aagccagtag agtgtcaaat tatgcacatt ggaattgaca tttgtcatca 89880 tattaaaatt cctgtgtagc cccatattga taggaattta accaggaagc ttgtctcagg 89940 actggagtca cacatttaat catataagca gacttgagga ctggagaccc taaaactgct 90000 tgcttgcact ggccatcatc tcccatcagg gtaggtggca gtcctttctc ctaaggagtt 90060 agtcttgttt atatgtattc aaggaaaaat acatcagtcc cttggaacta aaaggcatgc 90120 agtcctgagt ccccagatag gtgaatattg taacacatac ctttcccgaa atatgtttct 90180 gggatgctga gcagagaata gtctccttgt gatgtggatg ccgggtgttt ggccagcctc 90240 aatcaccagc tcaggtgcca ctgcctcaca cagtcactta gggtcattgg tttaggttat 90300 cattctacag cattttaaac tgacacattg tctggaccat gtgggttctt gaggactcat 90360 caaaacccgt tactaaaagc atgaatatca ggcgaaatag atagcaatgt gacattcgta 90420 tttatcccta agttccagtc taatgcagtg ccctggtatg tggagtgtag acagatgtgg 90480 gctaatcatg gaaggttccc tggaagttgt ggatattggt ttcgaattca gaaagctggg 90540 aaggatgtgg aaggctgaag gttggctttt ctagatttag ggcatgattt gaacaagtcc 90600 ttagaggtgg gaagggcagc acagggttgt tggcttggca agagtcaagg tgcaaagggt 90660 gacttggggt tcactggagg gaaacagaga tgagtgctct agaaggaagt tgagccttgt 90720 ggtgggtgac aggaaaccaa tgatgtaact tgtttttgac ctatctgggc cccaagtttg 90780 gatctgctat attaatataa aaaaggataa taatgataca ttcaaataat gctgaaaaat 90840 actaagatga aaatacctcc aacttcgtaa ttcaaaccat accattagga ttaggtgaac 90900 cacattccag gcgttttttt gcagagacag tgaaagggat ggctggctga aggaatgaat 90960 agatgaatgt tatatgcttt tgaacaatcg tcttttccat ttaattttct aattcaggag 91020 cagtaattat ccttgtgttg atcactgctg acgattttct atactgatag gtcctttccg 91080 ggggcttcca tctcttgcct tttaaatatg cttgcattga gattatctca ggtctttcca 91140 ttatgccatt actttcattt taaatcttct tgctctttca aatacacttt agttgtatct 91200 acagtgtttt aaaaacaatc tcattcagtg ttgtaatttc atctgtgggc tcttcctctg 91260 gatgaaatcc gtgttcctcc cagctgttcg gcagcatcag atggttgtga gggattctgt 91320 tgttctgttt tcttctaggc aaaggatgtg ccttcttttc atttgcagta gtctgctcac 91380 ccggaagcat gtcatttctt tgccacttgc ttgtaattca ctggctttgc acttgctctg 91440 atacagtaca ggtaactaat tgactccctc tgctgccaac ttggttttcc ttctgagcta 91500 tagcatcagg ctgtgtgttt tgtgttttct tgagattttg ttaaatatat ctggggtccc 91560 ttctacctgg ttggaactgg gattcccacc attcttgtgg ggatagaatc tcaggttaca 91620 cctatttccc caatcctctg tagccacaga agcttcatct tggccagctc tgttatcaga 91680 gtgcaggact tgggctgaaa tttcctcccc ttcctgattt tccttgacag tcctttccac 91740 tgctcctatc aatcaaaaga atgaaaaccc tcaacttgct gctttgcaga ttcaggtttt 91800 gtgcttcttt ctggcctctc ggggtggggc cgggttagca gcaaggctga gctgcccctc 91860 tttcttctga agccttcatg ggggcgagga gcacagggag agctcagtgc agggcctccc 91920 agtggccttc tcagagtggg tggaaaccca gcctggcact ggcagcgtgg caccagaagt 91980 atgaagtgta ggtgtaaagg tgatgtaaaa ggctagtagg ttttttggtt tttcattgtt 92040 tgagttttgg gcatagatga ctgtgaaggg cgaacactgc cgatggatct gaatgaattt 92100 gtagtatgtg caccacttcc aacttacggg atacccagct ttgacggctt tggacaaaca 92160 cactgaggcc aagatgtgct gagcttatca ggatcaggat caccaagcag ctgtaaaaac 92220 cctagcaagt gccttaagct gctgaaattt catattaatt gtctggtttg ttcatggtcc 92280 tagagtttga ggcagaaaag tcaggatcca agtcccttgg ttccaggcta cagctggaaa 92340 cagcatctcg gtgaactaaa gcaaccatat taggagtttt cctgctttag gagagtcccc 92400 agcatcggcg aggagggggc agcactctgg ctttccagga gcaaggggca ggatgcggcc 92460 gagggagagg ggctgtgttg aggaaaggag ggccgcaggc cctggggatg gtgtgaggct 92520 ccaaacatgt ccgagtcact tccctgggtg ggatgaggca gacagtgcca ccaccaggga 92580 cactttagtt agattagggt cttggaagtc acagaaggaa gtcagcagca gcaggctgga 92640 acttttctat gtataatcaa atggtttact ctgacaccgt tagcatgtaa caaacacaaa 92700 attttaaact aaggggaacc actaatggca tgtttccttt cctttcagat gataatttac 92760 tgtgatgtta gagaattcaa ggttgcagta aatggcgtac acagcctgga gtacaaacac 92820 agatttaaag agctcagcag tattgacacg ctggaaatta atggagacat ccacttactg 92880 gaagtaagga gctggtagcc tacctacaca gctgctacaa aaaccaaaat acagaatggc 92940 ttctgtgata ctggccttgc tgaaacgcat ctcactgtca ttctattgtt tatattgtta 93000 aaatgagctt gtgcaccatt agatcctgct gggtgttctc agtccttgcc atgaagtatg 93060 gtggtgtcta gcactgaatg gggaaactgg gggcagcaac acttatagcc agttaaagcc 93120 actctgccct ctctcctact ttggctgact cttcaagaat gccattcaac aagtatttat 93180 ggagtaccta ctataataca gtagctaaca tgtattgagc acagattttt tttggtaaaa 93240 ctgtgaggag ctaggatata tacttggtga aacaaaccag tatgttccct gttctcttga 93300 gcttcgactc ttctgtgctc tattgctgcg cactgctttt tctacaggca ttacatcaac 93360 tcctaagggg tcctctggga ttagttaagc agctattaaa tcacccgaag acactaattt 93420 acagaagaca caactccttc cccagtgatc actgtcataa ccagtgctct accgtatccc 93480 atcactgagg actgatgttg actgacatca ttttatcgta ataaacatgt ggctctatta 93540 gctgcaagct ttaccaagta attggcatga catctgagca cagaaattaa ggcaaaaaac 93600 caaagcaaaa caaatacatg gtgctgaaat taacttgatg ccaagcccaa ggcagctgat 93660 ttctgtgtat ttgaacttag ggcaaatcag agtctacaca gacgcctaca gaaagtttca 93720 ggaagaggca agatgcattc aatttgaaag atatttatgg gcaacaaagt aaggtcagga 93780 ttagacttca ggcattcata aggcaggcac tatcagaaag tgtacgccaa ctaagggacc 93840 cacaaagcag gcagaggtaa tgcagaaatc tgttttgttc ccatgaaatc accaatcaag 93900 gcctccgttc ttctaaagat tagtccatca tcattagcaa ctgagatcaa agcactcttc 93960 cactttacgt gattaaaatc aaacctgtat cagcaagtta aatggttcca tttctgtgat 94020 ttttctatta tttgagggga gttggcagaa gttccatgta tatgggatct ttacaggtca 94080 gatcttgtta caggaaattt caaaggtttg ggagtgggga gggaaaaaag ctcagtcagt 94140 gaggatcatt ttatcacatt agactggggc agaactctgc caggatttag gaatattttc 94200 agaacagatt ttagatatta tttctatcca tatattgaaa agaataccat tgtcaatctt 94260 atttttttaa aagtactcag tgtagaaatt gctagccctt aattcttttc cagcttttca 94320 tattaatgta tgcagagtct caccaagctc aaagacactg gttgggggtg gagggtgcca 94380 cagggaaagc tgtagaaggc aagaagactc gagaatcccc cagagttatt tttctccata 94440 aagaccatca gagtgcttaa ctgagctgtt ggagactgtg aggcatttag gaaaaaaata 94500 gcccactcac atcattcctt gtaagtctta agttcatttt cattttacgt ggaggaaaaa 94560 aatttaaaaa gctattagta tttattaatg aattttactg agacatttct tagaaatatg 94620 cacttctata ctagcaagct ctgtctctaa aatgcaagtt ggccttttgc ttgccacatt 94680 tctgcattaa acttctatat tagcttcaaa ggcttttaaa ctcaatgcga acattctacg 94740 ggatgttctt agatgccttt aaaaaggggg cagatctaat tttatttgaa ccctcacttt 94800 ccaacttcac catgacccag tactagagat tagggcactt caaagcattg aaaaaaatct 94860 actgatactt actttcttag acaagtagtt cttagttaac caccaatgga actgggttca 94920 ttctgaatcc tggaggagct tcctcgtgcc acccagtgtt tctgggccct ctgtgtgagc 94980 agccaggtat gagctgtttt agaagcagcg tgttgccttc atctctcccg tttcccaaaa 95040 gaacaaagga taaaggtgac agtcacactc ctgggttaaa aaaagcattc cagaaccact 95100 tctctttatg ggcacaacaa agaaacgaag gctgaagttc gcctacccaa aatgaaaagt 95160 aggctttaca gtcaaaagta cttctgttga ttgctaaata acttcatttt cttgaaatag 95220 agcaactttg agtgaaatct gcaacatgga taccatgtat ataagatact gctgtacaga 95280 agagttaagg cttacagtgc aaatgaggcg tcagctttgg gtgctaaaat taacaagtct 95340 aatattatta ccatcaatca ggaagagaat aataaatgtt taaacaaaca cagcagtctg 95400 tataaaaata ccgtgtatca tttactcttt ctgcagctct atacgatagg caggagaggc 95460 ttatgtggca gcacaagcca ggtggggatt ttgtaacgaa gtgataaaac atttgtaagt 95520 aatccaagta ggtgtattaa ggcaccaaaa gtaacatggc acccaacacc caaaaataaa 95580 aatatgaaat atgagtgtga actctgagta gagtatgaaa caccacagaa agtcttagaa 95640 atagctctgg agtggctctc ccaggacagt ttccagttgc tgaatagtct tttggcactg 95700 atgttctact tcttcacatt catctaaaaa aaaaaaaaaa aaaaatcaaa attaaaatct 95760 gagtcagtct gcctgcctcg gttctcatta gtttaattct taatgccttg cactttccag 95820 caatcattca atcaaaagag tgaaatgaag cacattaaca aagcaggagg cgccacggac 95880 cgcctccctc cacaccgctc cttccgcctt cattccttgc ccacaggctt gcactggaag 95940 ctgaataaga atccccaaaa ctcaaacttc ctagggatgc caccccttta gtagctcaca 96000 cctcccccct ccaagagcta agaaacaaag gagaatgtac ttttgtagct tagataagca 96060 atgaatcagt aaaggactga tctacttgct ccaccacccc tcccttaata ataacattta 96120 ctgttatttc ctgggcctaa gacttatgtt ccagaactgt cacagctccc catgtcacac 96180 ccactagctt gtgatctttg tcaaataact gaaatctttt aagcctctag tttcttcctt 96240 tgtaaaacag agataaaatg ttgtggtttt taagtgagat aatccaagta aagcacctaa 96300 catggagtag tgaatgaaca tcggttgcta ctaaaagtgg acatcctacc gcatccttaa 96360 tgccactagg catttccata caatctgggg accaaaactt caatcatata aatgtatgag 96420 gttaattaaa aacactactg taatctgctt gtatgatcac aaaccaccac aaaagaaaag 96480 atcgtgaaga ttacactgta aacggactct caaatgatca ggaggtggtc acttcgcaac 96540 ttgctccctc cacccaactc aaaacaggag ctcgagcctg cctgtatttg agactggagc 96600 tgcctgtatg aggactggat caactgctag tcacgttata tccaaatctg cattatcatt 96660 gggcacattt tcacagaatt ttactgaatt attccttaat tgtttaatgg ttgggaatag 96720 tttgggaatt accttccatc aactctgcta agaaaggaat ggattctggt agcaagacaa 96780 tataattctc ctttagtttt tcagccagtg ctaacacagt aatcaaagca gcaaatcgaa 96840 cctgaaaggg ataaaagagc aaagaaataa aaagtagtgt tactgtattt attatcttaa 96900 gagctgtact gacttgagac aagctctaac tttttaaaca ttagttcaca cgcgtttatt 96960 cacttcatta tgttcattaa gctttcatct tagaatacca gtttcaccat ttgggagctg 97020 tttgtaatat gtgcaacctt ataaatagtg ttttccaaac tgtgtcccag gactgcaaat 97080 ctttaatgtg aaatgtcttt ttataatctc ttcctttaaa aaaaaccaat aaaataaaat 97140 gccacatgca aactcaagtg tgtcaccaga ttttacttca ttggcgctcg ccagcccgcc 97200 aggctggcaa taaagtgcct ccagccacct ctggcaggtc tcctcaccca cagcccctga 97260 ctggtcacca ctatagttgt atgaggggcc aggacaatcg cttgggataa actcccatct 97320 cagcactgaa taaaaaacat tctgtgtcac aatatcctag ttttggggct ttaaaaacgt 97380 ctaggtgttc ctcacatgcc ttgtctataa taaggaaagc aagcagtagt tgggtattgt 97440 tagcttttga aacaaaagcc ctactggtct tctaattttg gatattttaa ttaaagaata 97500 tctggacagt acaaagtgaa ttattaaaaa accatttgta actacctaga ttcaatcagg 97560 atttccttga tttgtgcaaa gtaaaatatt acaataaatt tgatactgct acttgtataa 97620 aaacctatgg tttaaaatgt gggggttcat cataatagtc tcattgttag catatcctaa 97680 taaagaattt gaactaataa atcctattaa taaaattctg ctttggtctg ttatagccag 97740 taaagttcta atacaatcat tagtttgaga aatggtgact cattgctaaa acagtttgaa 97800 atttgtaaca cttgggtgtc aaattttgac ttccactcaa cctacccatg ttttatttcc 97860 actgccacca cttactcaac aagatcataa gcctagtatc tataaacaac agaatgtatt 97920 gctctaactc aaaagactat agtgtggata aattcaatgc atttctctct ggagcacaat 97980 gacatttcaa tagcacttaa aaaagaagga attacttcaa atctttgtta tttaaaagta 98040 tttagaaagt attttagtac ttctgcccaa cgcaccattg gggtggggat agggcattgc 98100 tattctttac aaatagccta taagtaaaaa acaaaatttt cttaggcaca aatttctgcc 98160 taatacaaaa gaccagacct ctagtactgg atgacaaata gcaatgttct tccctgccag 98220 tttactaggg ggcctacatc tgtgaccacc tgcaggctgt ttaggctatg cagtgaaaag 98280 atgcagtttc agtacttgtc acgcagttcc taaccttagg cgaggagtct ctcgtcttta 98340 gcagaatctg gtagttcagt ggtttccaaa gagagtcatc cgccatggcc actgaaaact 98400 gtgcgatgca tggtatcagg tgctttgtca cccgttcctg gaatttctct tctcccccaa 98460 gcctgttttc cagctaggaa gagtaagaca aagactttga acaacaagtc tcatttcttt 98520 cttctgtttg aaaaaatgtc caacatacaa atattttact atctttcatg atattagcag 98580 gttcaaaaac caggcattat tctaatactc tctagggcaa atgtattgcc ttctagaact 98640 caaatggaat ctcataccct ttatcatcgc ccctttctct ccagcagaac atctcagagg 98700 agctctttgc tccagaggac agccatgctc tgacacgttc tcagtgaggc ccagttaaaa 98760 caaatgaata cattaaccat gacagcttat atcatgtctg tcttttgagc agtttaaaaa 98820 ataaaaaata aaaaataact cagggccagg catggtggct cacgcctgta atcccagcag 98880 tttgggaggc caaggtgggt ggatcacttg aggtcaggag ttcgagacca gcctggccaa 98940 catggcaaaa cctcatccct actaaaaata caaaaattag ccaggtgtgg aggcgggcgc 99000 ctgtgatccc agctattcgg gaggctgagg cacaagaatt gcttgaaccc gggaggtgga 99060 ggttgcagcg agccgagatt gcaccactgc actccagcct gggtgacaga gcaagaccct 99120 gtctcaaaac aacaaaacaa aactcaaatt ccacaatgaa gttatatctt tgaaaaaaca 99180 attttcaaat aaaacatttc attaaaaaga ccagaaaaaa caaccttaca aagaaaaatc 99240 ctagcaagct gtcatttgag cagatctaaa acctgccaag ctcgaacagt gatggcttcc 99300 tcagcaacga aagatgattc tgtttggtta cctgatccac cagaggcatc atcaaggctc 99360 ctgctctctc tttacttata aaatgctggg tatcaaaaag gaagattttg tataaacagt 99420 tcaaaataaa ctgcaacagc aagcagcact tttcagggtc attttcagag tcaaaaaatg 99480 cttcatctgt agacgtggga agagtaaaaa tgaaaaaaca ctgaacttaa ccatttaatc 99540 tccaatgttt acattgaaat cactattaaa ataactaaat cagaagagtc taaaatgatc 99600 tagaaatcat aatcaggacg aaggcagaac acaatggatg gtctctcgaa gaatgattcc 99660 ttcttttaga gttaagattc taacactcac tctggcaagt taaattccct caactgtcaa 99720 gtgggtcacg tattagcatt agagaataaa ctaatcttaa tttttgcgtt ttaaagttac 99780 ttccagtaac tgacagtaac ggccatttac tttattcttt ctcccaagtg aggtgactta 99840 taacattcgc tcatcatgct aaaacaacac ttcactgtct gacaacaatg aagtaaaaaa 99900 ttcaccctcc ttagcttagg acttaagaac ctctaaaatc ttgcttccaa gcactagctt 99960 gtgtcttact ggtaccttgt ataaggcaca caggacaagg gtgacagctg aactgaagcg 100020 accacccacc tgttttggag atgttcacct ggtccaaggt gtcagcaaaa ggcttcacta 100080 agtggccggc aaacagagta aaaagccctt tcagcttttc agcaatgcaa tctgccaagt 100140 tgtaaaatgt caacaacctg tcctttgggg catcttctgt tttagcccaa tcaaacagct 100200 gaaaggataa gacagtatta gtttcttcga catcttgtca cttaaatctg agcacaaaag 100260 agaggaagag gaagaaagcg tcaccttgaa gaacaggggc ctgaatgtga cctcggaaag 100320 tttgacaacc atggctacta gacagtcaat gatacaattt tccgtttttc caacttcctc 100380 cagatcgttc tgaaaacaga agagcccatt tattagagtg ctgatacctg actgtaaatt 100440 attttggcaa gtaccactgt tacacggcta gattgttctc ggactcttca ataggtggat 100500 aacagcttta ggatttggag gagtgaacct gagcttacct cagagtgctg ggctcggaag 100560 tccagggcct ccaggaaaaa ggcggttagc tgagactgat gggaggtgag ctcttccttc 100620 ttcatcgccc caatatgctc ttgcaagatg ctcataaacg gacccatgtg attctaccaa 100680 taacacagga aaaagatgtg ccattttcaa atgattccta gagttcagcg gtgtgtattt 100740 ttaaaaacta aatcttcttc tttaagtcaa agtttacaca ttgcagtacc acctctccct 100800 tctccaaagt cttaataccc aataagatct aaccttccag ttcttctcaa tctgcttgta 100860 agtttttttg atggcgggca acaggactcg gggtgcaagt gtggtagcca gtgtcttttt 100920 aagagatgtg agacggatat tagcctgtga cgcagaaccc atttcactag tgattttctc 100980 cagatgaatc acctacagga atataaaaaa agtgatcagg gccactgcag atcttcgctg 101040 acaaacacac acttacagag aggcttcatg atgaggtact agtgtttgga aaatgcttag 101100 cactttttaa ctacacacag agttcctttt aaagtcagcc ctaaacgtca gtggataaaa 101160 ctgggcagac acctcttgcc caacttgcga tcagggacga aggccgatgg tagacgcaga 101220 cgcacacaca gcacccagac agatgatttt cttagaggac aggaatgcaa gggaccacgg 101280 caagagtcaa gttgctaaaa aactgagaaa gctcctcaga gcacaggccc ctttctctga 101340 gaaggctact tttaaaccct ggctgtggtg taagtgaagc ggtttaatca tttgccccat 101400 ggtaatgaag gctcctaacc ttgtaaatgg caaatgatca acacaatgga acagccaggt 101460 ctcaacactc ttgagcatct tcaatcataa ataccactgg cccctagcgt gttgacagga 101520 aaccgctgac gtgcaataca aaaattctgc tttgcaagat gccttaggat taaacctctc 101580 acagtagaaa cagggcccat caatttccac aagtaataaa aggcggctct accagcccaa 101640 ctccaaagat ctcacagaag aaaaaaaagc cagaatacat tccgcacaat taaagaagag 101700 aagcatctcg ctaaaaagtg acccccatat caatttcaag attaagtggc aaggatgatg 101760 gaagagaaaa agtacacatt taataaaagc aagcacatct cttcagaaat aagactcctt 101820 tctgtcaaac ggaaactaac ccttaaagaa aaaacaaaat cactacattt gtgatctttt 101880 accttcccca gccaccctgc gtagcatgtc gtggctatcg tggctcacct gggagagaat 101940 gccttccaga taggggctga tgaagtgcgg gagagtctcc acaaccttct gcagagcagc 102000 caaggcactg agcaggtaga cctcgctgga gaccagctcg ctggtgttct tcattgttgt 102060 cagcaacgat ggcatcaggc tagaaacaaa gtaagagctt tagaagaact tgaagcagaa 102120 acagaggcta gggaatggag tagagggcat tatgaaaaaa accagcaaac tgtgcctatt 102180 acatcgctat ctgcctcata gcctaaaaag cagtgtctat acattttatg tggctaagca 102240 caagaaatct cccagtgcta acagtatgga cacaacagta atttaaaaaa taacaatgtc 102300 tttcattaac tgaacactta ctatgtgtca ggcactatgc aaaactcctt gcaagcactg 102360 ccctacagaa atcctatgag gtagatactg tctctgtttt atagacagca aagctctaac 102420 aggttaagga acatactggc tgtacagtaa ggaactacca cagccaggag cttctaactt 102480 ccaaatttgg cagcagaagg cagctttggc cttgcctaac tgggtgggcc cctctgccaa 102540 gaaccttcac ccactgcttt ttgactatac tagacaaaag gaaggaagaa tggaggacga 102600 ttaacactgc aaagcagtgc atctgaagat aaacgggaag gctgcatctt tctgtttgaa 102660 gattaattat ttttattatt atttctttaa gagacagggt ctcactctgt tgcccaggct 102720 acagtgcagt ggtgcagtca tagctcactg cagcctcaaa ctcctgggct caaatgatct 102780 ccctgccttg gcctcccaaa gtgctgggat cacagccgtg agccaccaca ccctgcaaga 102840 tcaattcttt aacaaattcc aattttatgc aacgtctact cagaggaaaa aaaaaaaaag 102900 tcaccaaagt gttatttttc aatgtgtgcc aggcggtaac agctcctgtt ccaagtctcc 102960 ggccgcatac ctgggaagct gggggatggc cagcgcctcc agggtggagg tcacctctgc 103020 tatgcacagc agcgcgcttc ccaagacatt cttctcctcc tttctctctg gagcaatcag 103080 tttcacagca gtgctcagca ctgggacaaa aggatctgga ttttctgcac caaaattctt 103140 gcataaaagc tttaaggtat acaacgctgt ctgtctgttg attgcttgtt cttcttcccc 103200 ttcctttttc ttacgctgca caatggccaa aaggtctgga accagtttta ggaaacgggt 103260 aacctgaagg ggacagccag aatccccaaa tcattaaagc tgcaaaaaat gtttgtccat 103320 tttcccattg tcacagcttg agattgtcta aatggaaatc agactcgggg gtcctgagtc 103380 acacagtcat gctaagcgat gtgcatgttc tagccagtgt ttcacttata caaagcaccc 103440 actgatctgg agtaaaaggg acttagaact atgctaaggc taaggccacg taagctctgt 103500 agtaagcaag aattccacta ggctgaaatt ccattctaag agctcttaca acacacatat 103560 attcccgtta gaattaacgt cacattttaa aacatgtcat ggtattatat tcagataata 103620 atatacttca atttgaaatt gtaccactag agaaattgaa gggagttaaa tgcagctctt 103680 tgataaagca aagtacagta aatgggtgtg tcctgggtct tcactcacta ttgtcttctt 103740 ccaggatata ttttgctgca gcttgttatt caaaaggtcc agcgctttgc ggcgaacaga 103800 tggcagggga ttgcccacca gccctctgat cacaggaatg aatgtctctg tgggcagcaa 103860 ggcattgacc taaagagaaa ttttatattt aacatgaaaa gaaaaacaaa ttaaaaaaaa 103920 aatcaacttc aattaagaca gactgctgtc cactgcacac ctccaggcac caggcacttc 103980 cacacacatt ttcttattta attcttaaaa taacctttca ggtaggcatt accaaccaca 104040 cattatcgaa caaaacaaaa gcctgatgtc aggaggaagt gccaaaggca tgcagctaaa 104100 tgactgagct agatttgaat cagcaatcct aacttcgagg ccagtgatat gtatgtaata 104160 tacttcatac ttttatttta ttccacttga ataaagtaga acagtatata ttatatgact 104220 taattattaa aatatacgag gtacatgttc tcataactgg taaggaaaca attttttcca 104280 gacaaatcta tttctagtca tcaagagatt gttttctaag aaaaatctga gcttcattat 104340 attcataaaa ggaattgcta agtttattct taaaaacttt acataatttc acaataattt 104400 aaaaaacagc aacaaaacag taattccagg gagaaatgaa cacctacctt atctaacagg 104460 tcgtaagctt tactaaggag cgcgcgccag aacttcacgg tgagtttgtc tgcgttcctt 104520 tccatggact gtgcaactgc actgatatag ccgagaacgg tctccagcaa cctgaaacac 104580 agaggctcgc tcagcaaacg gcagctgaag aaactcagag aacttgttca tgtctacctt 104640 atgctaaatg tttcaagtag aaagacgagt taaataattc tgtactaaat tatttcaaaa 104700 actactcgga aagaaaggaa atgagggatt attgccatag acagagatca tcaagaagta 104760 actaggcgct tctgtgcaga agcatcgacc tcgctcagac tctgtgaggt gctgaataag 104820 caacagatgc tgaaagcgtt taaggaactc actcatatct agctcatgct cagtggatct 104880 cactgggctg tccaagtggg gtgttcaggg agttatggcc ctaggttaat ggcaggtgtg 104940 tgcgtgcaca cacacacagg cacacacacg cacacataca catgcacaca caccatacac 105000 catttatata aagagaaata ttaatagaaa tgaacatata acccacttct ttcacattat 105060 taggagacaa aaaaaaagac tacaaacttc aaataacttg taattagaaa agcacacacc 105120 aaattccaac acagctgcca ctggagatcc ccccactgct gccagcctga ggggggagct 105180 agagggaaga gtggagacag aagttgacac cgcacagcag aggaggggag aagggggcgc 105240 agacaaaatc agctccaaaa acgaaagtcc tacgcatagc gctacaagtc agcccacagg 105300 actggaactc agcagctcac attcctggct gcagggcagg cactttccag tggaaggggc 105360 aggacagtgg ccctgggaat gccatgcatc tgaaaaggag gtacacagca aggccaggag 105420 gcaaaccccg aggacatggg agagaaagga aaattcctgc acccaaatat ataatggcag 105480 catatggatt agaatccacg gaataaagaa ttcatgagcc catagaaatc agggccagat 105540 tgagacacta aacagatact gcaactcaat acaatacaca gacttgacat ggatcatgat 105600 gcagaaacac atgcggtgta aaggacagtg ttgggataat tagggagact ggagtatgaa 105660 ctgtagatta catcactgga ttggatcaat gttaaatttt ctgaatttga tcaatgtact 105720 gtggttttat aagaacatct cttattctta gagacataat gtatatgatt tactttcaaa 105780 tggctcagag aaaaaaccct acatagggag aacgctaagg caaatgtggc agaaagtatt 105840 atcaaatggt gaacctggtt gtaaagagta tatgaatttt ctgtactgtt tttccaggtt 105900 ttctataagt ttgaagtcat ttccaaataa aaagtaaaaa aagaaaagga aacatacctc 105960 tcttcaaggc cttttaaaat ctcaggacca ccactctcaa ctacctaatt tttaaagaag 106020 acgtcattag aacggtatgg aagtcaataa taaaagtcat ttcaagtcag ttcaatgaaa 106080 ctcggaccat tcactgaaac cttccacagc aactgttttc tgacattaca atttaatcag 106140 gttcatagca tcttcattat actgtagtaa ctctatttct cttaatttat tttaattata 106200 ttctactggt agtatctaaa aagtactaca atggttcaga aaaatacagc aatcaacact 106260 caattagcac taccgaattc tatgacatgc tgatctggtg agctcacata tcctttgttg 106320 agaagttaaa cattacagat tcagctggaa tcccccaagt actgctcctt ggtcctattc 106380 tccctctacc ccaagcccca caaacaaaac catcatccca aatctgcttc caaatgtttc 106440 aaacactaca tatcacggaa caacatgttt ttctggaaac atatttttga gatctatgca 106500 tggtgactta tgttctagtt ccttcatttt aactgcatat gatattcctc tataaatacc 106560 acttatctat ccatttgcct ctgttgttag atgtttagtt tatgtccatt ttttcccctt 106620 ttactaataa tgctagagaa gaacattttt atgtcccttt gatcatcttg ggaagttttt 106680 acagcatata tacctaagga agggaatgac cagatcacag gaattactgg aactttcaac 106740 ctcatg 106746 2 5408 DNA Homo sapiens CDS 201..1151 polyA_signal 1773..1778 AATAAA 2 agccgcccac ggacgccaga gccgggaacc ctgacggcac ttagctgctg acaaacaacc 60 tgctccgtgg agcgcctgaa acaccagtct ttggggccag tgcctcagtt tcaatccagg 120 taacctttaa atgaaacttg cctaaaatct taggtcatac acagaagaga ctccaatcga 180 caagaagctg gaaaagaatg atg ttg tcc tta aac aac cta cag aat atc atc 233 Met Leu Ser Leu Asn Asn Leu Gln Asn Ile Ile 1 5 10 tat aac ccg gta atc ccg tat gtt ggc acc att ccc gat cag ctg gat 281 Tyr Asn Pro Val Ile Pro Tyr Val Gly Thr Ile Pro Asp Gln Leu Asp 15 20 25 cct gga act ttg att gtg ata tgt ggg cat gtt cct agt gac gca gac 329 Pro Gly Thr Leu Ile Val Ile Cys Gly His Val Pro Ser Asp Ala Asp 30 35 40 aga ttc cag gtg gat ctg cag aat ggc agc agt gtg aaa cct cga gcc 377 Arg Phe Gln Val Asp Leu Gln Asn Gly Ser Ser Val Lys Pro Arg Ala 45 50 55 gat gtg gcc ttt cat ttc aat cct cgt ttc aaa agg gcc ggc tgc att 425 Asp Val Ala Phe His Phe Asn Pro Arg Phe Lys Arg Ala Gly Cys Ile 60 65 70 75 gtt tgc aat act ttg ata aat gaa aaa tgg gga cgg gaa gag atc acc 473 Val Cys Asn Thr Leu Ile Asn Glu Lys Trp Gly Arg Glu Glu Ile Thr 80 85 90 tat gac acg cct ttc aaa aga gaa aag tct ttt gag atc gtg att atg 521 Tyr Asp Thr Pro Phe Lys Arg Glu Lys Ser Phe Glu Ile Val Ile Met 95 100 105 gtg cta aag gac aaa ttc cag gtg gct gta aat gga aaa cat act ctg 569 Val Leu Lys Asp Lys Phe Gln Val Ala Val Asn Gly Lys His Thr Leu 110 115 120 ctc tat ggc cac agg atc ggc cca gag aaa ata gac act ctg ggc att 617 Leu Tyr Gly His Arg Ile Gly Pro Glu Lys Ile Asp Thr Leu Gly Ile 125 130 135 tat ggc aaa gtg aat att cac tca att ggt ttt agc ttc agc tcg gac 665 Tyr Gly Lys Val Asn Ile His Ser Ile Gly Phe Ser Phe Ser Ser Asp 140 145 150 155 tta caa agt acc caa gca tct agt ctg gaa ctg aca gag ata agt aga 713 Leu Gln Ser Thr Gln Ala Ser Ser Leu Glu Leu Thr Glu Ile Ser Arg 160 165 170 gaa aat gtt cca aag tct ggc acg ccc cag ctt agc ctg cca ttc gct 761 Glu Asn Val Pro Lys Ser Gly Thr Pro Gln Leu Ser Leu Pro Phe Ala 175 180 185 gca agg ttg aac acc ccc atg ggc cct gga cga act gtc gtc gtt aaa 809 Ala Arg Leu Asn Thr Pro Met Gly Pro Gly Arg Thr Val Val Val Lys 190 195 200 gga gaa gtg aat gca aat gcc aaa agc ttt aat gtt gac cta cta gca 857 Gly Glu Val Asn Ala Asn Ala Lys Ser Phe Asn Val Asp Leu Leu Ala 205 210 215 gga aaa tca aag gat att gct cta cac ttg aac cca cgc ctg aat att 905 Gly Lys Ser Lys Asp Ile Ala Leu His Leu Asn Pro Arg Leu Asn Ile 220 225 230 235 aaa gca ttt gta aga aat tct ttt ctt cag gag tcc tgg gga gaa gaa 953 Lys Ala Phe Val Arg Asn Ser Phe Leu Gln Glu Ser Trp Gly Glu Glu 240 245 250 gag aga aat att acc tct ttc cca ttt agt cct ggg atg tac ttt gag 1001 Glu Arg Asn Ile Thr Ser Phe Pro Phe Ser Pro Gly Met Tyr Phe Glu 255 260 265 atg ata att tac tgt gat gtt aga gaa ttc aag gtt gca gta aat ggc 1049 Met Ile Ile Tyr Cys Asp Val Arg Glu Phe Lys Val Ala Val Asn Gly 270 275 280 gta cac agc ctg gag tac aaa cac aga ttt aaa gag ctc agc agt att 1097 Val His Ser Leu Glu Tyr Lys His Arg Phe Lys Glu Leu Ser Ser Ile 285 290 295 gac acg ctg gaa att aat gga gac atc cac tta ctg gaa gta agg agc 1145 Asp Thr Leu Glu Ile Asn Gly Asp Ile His Leu Leu Glu Val Arg Ser 300 305 310 315 tgg tag cctacctaca cagctgctac aaaaaccaaa atacagaatg gcttctgtga 1201 Trp * tactggcctt gctgaaacgc atctcactgt cattctattg tttatattgt taaaatgagc 1261 ttgtgcacca ttagatcctg ctgggtgttc tcagtccttg ccatgaagta tggtggtgtc 1321 tagcactgaa tggggaaact gggggcagca acacttatag ccagttaaag ccactctgcc 1381 ctctctccta ctttggctga ctcttcaaga atgccattca acaagtattt atggagtacc 1441 tactataata cagtagctaa catgtattga gcacagattt tttttggtaa aactgtgagg 1501 agctaggata tatacttggt gaaacaaacc agtatgttcc ctgttctctt gagcttcgac 1561 tcttctgtgc tctattgctg cgcactgctt tttctacagg cattacatca actcctaagg 1621 ggtcctctgg gattagttaa gcagctatta aatcacccga agacactaat ttacagaaga 1681 cacaactcct tccccagtga tcactgtcat aaccagtgct ctaccgtatc ccatcactga 1741 ggactgatgt tgactgacat cattttatcg taataaacat gtggctctat tagctgcaag 1801 ctttaccaag taattggcat gacatctgag cacagaaatt aaggcaaaaa accaaagcaa 1861 aacaaataca tggtgctgaa attaacttga tgccaagccc aaggcagctg atttctgtgt 1921 atttgaactt agggcaaatc agagtctaca cagacgccta cagaaagttt caggaagagg 1981 caagatgcat tcaatttgaa agatatttat gggcaacaaa gtaaggtcag gattagactt 2041 caggcattca taaggcaggc actatcagaa agtgtacgcc aactaaggga cccacaaagc 2101 aggcagaggt aatgcagaaa tctgttttgt tcccatgaaa tcaccaatca aggcctccgt 2161 tcttctaaag attagtccat catcattagc aactgagatc aaagcactct tccactttac 2221 gtgattaaaa tcaaacctgt atcagcaagt taaatggttc catttctgtg atttttctat 2281 tatttgaggg gagttggcag aagttccatg tatatgggat ctttacaggt cagatcttgt 2341 tacaggaaat ttcaaaggtt tgggagtggg gagggaaaaa agctcagtca gtgaggatca 2401 ttttatcaca ttagactggg gcagaactct gccaggattt aggaatattt tcagaacaga 2461 ttttagatat tatttctatc catatattga aaagaatacc attgtcaatc ttattttttt 2521 aaaagtactc agtgtagaaa ttgctagccc ttaattcttt tccagctttt catattaatg 2581 tatgcagagt ctcaccaagc tcaaagacac tggttggggg tggagggtgc cacagggaaa 2641 gctgtagaag gcaagaagac tcgagaatcc cccagagtta tttttctcca taaagaccat 2701 cagagtgctt aactgagctg ttggagactg tgaggcattt aggaaaaaaa tagcccactc 2761 acatcattcc ttgtaagtct taagttcatt ttcattttac gtggaggaaa aaaatttaaa 2821 aagctattag tatttattaa tgaattttac tgagacattt cttagaaata tgcacttcta 2881 tactagcaag ctctgtctct aaaatgcaag ttggcctttt gcttgccaca tttctgcatt 2941 aaacttctat attagcttca aaggctttta aactcaatgc gaacattcta cgggatgttc 3001 ttagatgcct ttaaaaaggg ggcagatcta attttatttg aaccctcact ttccaacttc 3061 accatgaccc agtactagag attagggcac ttcaaagcat tgaaaaaaat ctactgatac 3121 ttactttctt agacaagtag ttcttagtta accaccaatg gaactgggtt cattctgaat 3181 cctggaggag cttcctcgtg ccacccagtg tttctgggcc ctctgtgtga gcagccaggt 3241 atgagctgtt ttagaagcag cgtgttgcct tcatctctcc cgtttcccaa aagaacaaag 3301 gataaaggtg acagtcacac tcctgggtta aaaaaagcat tccagaacca cttctcttta 3361 tgggcacaac aaagaaacga aggctgaagt tcgcctaccc aaaatgaaaa gtaggcttta 3421 cagtcaaaag tacttctgtt gattgctaaa taacttcatt ttcttgaaat agagcaactt 3481 tgagtgaaat ctgcaacatg gataccatgt atataagata ctgctgtaca gaagagttaa 3541 ggcttacagt gcaaatgagg cgtcagcttt gggtgctaaa attaacaagt ctaatattat 3601 taccatcaat caggaagaga ataataaatg tttaaacaaa cacagcagtc tgtataaaaa 3661 taccgtgtat catttactct ttctgcagct ctatacgata ggcaggagag gcttatgtgg 3721 cagcacaagc caggtgggga ttttgtaacg aagtgataaa acatttgtaa gtaatccaag 3781 taggtgtatt aaggcaccaa aagtaacatg gcacccaaca cccaaaaata aaaatatgaa 3841 atatgagtgt gaactctgag tagagtatga aacaccacag aaagtcttag aaatagctct 3901 ggagtggctc tcccaggaca gtttccagtt gctgaatagt cttttggcac tgatgttcta 3961 cttcttcaca ttcatctaaa aaaaaaaaaa aaaaaaatca aaattaaaat ctgagtcagt 4021 ctgcctgcct cggttctcat tagtttaatt cttaatgcct tgcactttcc agcaatcatt 4081 caatcaaaag agtgaaatga agcacattaa caaagcagga ggcgccacgg accgcctccc 4141 tccacaccgc tccttccgcc ttcattcctt gcccacaggc ttgcactgga agctgaataa 4201 gaatccccaa aactcaaact tcctagggat gccacccctt tagtagctca cacctccccc 4261 ctccaagagc taagaaacaa aggagaatgt acttttgtag cttagataag caatgaatca 4321 gtaaaggact gatctacttg ctccaccacc cctcccttaa taataacatt tactgttatt 4381 tcctgggcct aagacttatg ttccagaact gtcacagctc cccatgtcac acccactagc 4441 ttgtgatctt tgtcaaataa ctgaaatctt ttaagcctct agtttcttcc tttgtaaaac 4501 agagataaaa tgttgtggtt tttaagtgag ataatccaag taaagcacct aacatggagt 4561 agtgaatgaa catcggttgc tactaaaagt ggacatccta ccgcatcctt aatgccacta 4621 ggcatttcca tacaatctgg ggaccaaaac ttcaatcata taaatgtatg aggttaatta 4681 aaaacactac tgtaatctgc ttgtatgatc acaaaccacc acaaaagaaa agatcgtgaa 4741 gattacactg taaacggact ctcaaatgat caggaggtgg tcacttcgca acttgctccc 4801 tccacccaac tcaaaacagg agctcgagcc tgcctgtatt tgagactgga gctgcctgta 4861 tgaggactgg atcaactgct agtcacgtta tatccaaatc tgcattatca ttgggcacat 4921 tttcacagaa ttttactgaa ttattcctta attgtttaat ggttgggaat agtttgggaa 4981 ttaccttcca tcaactctgc taagaaagga atggattctg gtagcaagac aatataattc 5041 tcctttagtt tttcagccag tgctaacaca gtaatcaaag cagcaaatcg aacctgaaag 5101 ggataaaaga gcaaagaaat aaaaagtagt gttactgtat ttattatctt aagagctgta 5161 ctgacttgag acaagctcta actttttaaa cattagttca cacgcgttta ttcacttcat 5221 tatgttcatt aagctttcat cttagaatac cagtttcacc atttgggagc tgtttgtaat 5281 atgtgcaacc ttataaatag tgttttccaa actgtgtccc aggactgcaa atctttaatg 5341 tgaaatgtct ttttataatc tcttccttta aaaaaaacca ataaaataaa atgccacatg 5401 caaactc 5408 3 5534 DNA Homo sapiens misc_feature 201..203 ATG 3 agccgcccac ggacgccaga gccgggaacc ctgacggcac ttagctgctg acaaacaacc 60 tgctccgtgg agcgcctgaa acaccagtct ttggggccag tgcctcagtt tcaatccagg 120 taacctttaa atgaaacttg cctaaaatct taggtcatac acagaagaga ctccaatcga 180 caagaagctg gaaaagaatg atg ttg tcc tta aac aac cta cag aat atc atc 233 Met Leu Ser Leu Asn Asn Leu Gln Asn Ile Ile 1 5 10 tat aac ccg gta atc ccg tat gtt ggc acc att ccc gat cag ctg gat 281 Tyr Asn Pro Val Ile Pro Tyr Val Gly Thr Ile Pro Asp Gln Leu Asp 15 20 25 cct gga act ttg att gtg ata tgt ggg cat gtt cct agt gac gca gac 329 Pro Gly Thr Leu Ile Val Ile Cys Gly His Val Pro Ser Asp Ala Asp 30 35 40 aga ttc cag gtg gat ctg cag aat ggc agc agt gtg aaa cct cga gcc 377 Arg Phe Gln Val Asp Leu Gln Asn Gly Ser Ser Val Lys Pro Arg Ala 45 50 55 gat gtg gcc ttt cat ttc aat cct cgt ttc aaa agg gcc ggc tgc att 425 Asp Val Ala Phe His Phe Asn Pro Arg Phe Lys Arg Ala Gly Cys Ile 60 65 70 75 gtt tgc aat act ttg ata aat gaa aaa tgg gga cgg gaa gag atc acc 473 Val Cys Asn Thr Leu Ile Asn Glu Lys Trp Gly Arg Glu Glu Ile Thr 80 85 90 tat gac acg cct ttc aaa aga gaa aag tct ttt gag atc gtg att atg 521 Tyr Asp Thr Pro Phe Lys Arg Glu Lys Ser Phe Glu Ile Val Ile Met 95 100 105 gtg cta aag gac aaa ttc cag gtg gct gta aat gga aaa cat act ctg 569 Val Leu Lys Asp Lys Phe Gln Val Ala Val Asn Gly Lys His Thr Leu 110 115 120 ctc tat ggc cac agg atc ggc cca gag aaa ata gac act ctg ggc att 617 Leu Tyr Gly His Arg Ile Gly Pro Glu Lys Ile Asp Thr Leu Gly Ile 125 130 135 tat ggc aaa gtg aat att cac tca att ggt ttt agc ttc agc tcg gac 665 Tyr Gly Lys Val Asn Ile His Ser Ile Gly Phe Ser Phe Ser Ser Asp 140 145 150 155 tta caa agt acc caa gca tct agt ctg gaa ctg aca gag ata agt aga 713 Leu Gln Ser Thr Gln Ala Ser Ser Leu Glu Leu Thr Glu Ile Ser Arg 160 165 170 gaa aat gtt cca aag tct ggc acg ccc cag ctt cct agt aat aga gga 761 Glu Asn Val Pro Lys Ser Gly Thr Pro Gln Leu Pro Ser Asn Arg Gly 175 180 185 gga gac att tct aaa atc gca ccc aga act gtc tac acc aag agc aaa 809 Gly Asp Ile Ser Lys Ile Ala Pro Arg Thr Val Tyr Thr Lys Ser Lys 190 195 200 gat tcg act gtc aat cac act ttg act tgc acc aaa ata cca cct atg 857 Asp Ser Thr Val Asn His Thr Leu Thr Cys Thr Lys Ile Pro Pro Met 205 210 215 aac tat gtg tca aag agc ctg cca ttc gct gca agg ttg aac acc ccc 905 Asn Tyr Val Ser Lys Ser Leu Pro Phe Ala Ala Arg Leu Asn Thr Pro 220 225 230 235 atg ggc cct gga cga act gtc gtc gtt aaa gga gaa gtg aat gca aat 953 Met Gly Pro Gly Arg Thr Val Val Val Lys Gly Glu Val Asn Ala Asn 240 245 250 gcc aaa agc ttt aat gtt gac cta cta gca gga aaa tca aag gat att 1001 Ala Lys Ser Phe Asn Val Asp Leu Leu Ala Gly Lys Ser Lys Asp Ile 255 260 265 gct cta cac ttg aac cca cgc ctg aat att aaa gca ttt gta aga aat 1049 Ala Leu His Leu Asn Pro Arg Leu Asn Ile Lys Ala Phe Val Arg Asn 270 275 280 tct ttt ctt cag gag tcc tgg gga gaa gaa gag aga aat att acc tct 1097 Ser Phe Leu Gln Glu Ser Trp Gly Glu Glu Glu Arg Asn Ile Thr Ser 285 290 295 ttc cca ttt agt cct ggg atg tac ttt gag atg ata att tac tgt gat 1145 Phe Pro Phe Ser Pro Gly Met Tyr Phe Glu Met Ile Ile Tyr Cys Asp 300 305 310 315 gtt aga gaa ttc aag gtt gca gta aat ggc gta cac agc ctg gag tac 1193 Val Arg Glu Phe Lys Val Ala Val Asn Gly Val His Ser Leu Glu Tyr 320 325 330 aaa cac aga ttt aaa gag ctc agc agt att gac acg ctg gaa att aat 1241 Lys His Arg Phe Lys Glu Leu Ser Ser Ile Asp Thr Leu Glu Ile Asn 335 340 345 gga gac atc cac tta ctg gaa gta agg agc tgg tag cctacctaca 1287 Gly Asp Ile His Leu Leu Glu Val Arg Ser Trp * 350 355 cagctgctac aaaaaccaaa atacagaatg gcttctgtga tactggcctt gctgaaacgc 1347 atctcactgt cattctattg tttatattgt taaaatgagc ttgtgcacca ttagatcctg 1407 ctgggtgttc tcagtccttg ccatgaagta tggtggtgtc tagcactgaa tggggaaact 1467 gggggcagca acacttatag ccagttaaag ccactctgcc ctctctccta ctttggctga 1527 ctcttcaaga atgccattca acaagtattt atggagtacc tactataata cagtagctaa 1587 catgtattga gcacagattt tttttggtaa aactgtgagg agctaggata tatacttggt 1647 gaaacaaacc agtatgttcc ctgttctctt gagcttcgac tcttctgtgc tctattgctg 1707 cgcactgctt tttctacagg cattacatca actcctaagg ggtcctctgg gattagttaa 1767 gcagctatta aatcacccga agacactaat ttacagaaga cacaactcct tccccagtga 1827 tcactgtcat aaccagtgct ctaccgtatc ccatcactga ggactgatgt tgactgacat 1887 cattttatcg taataaacat gtggctctat tagctgcaag ctttaccaag taattggcat 1947 gacatctgag cacagaaatt aaggcaaaaa accaaagcaa aacaaataca tggtgctgaa 2007 attaacttga tgccaagccc aaggcagctg atttctgtgt atttgaactt agggcaaatc 2067 agagtctaca cagacgccta cagaaagttt caggaagagg caagatgcat tcaatttgaa 2127 agatatttat gggcaacaaa gtaaggtcag gattagactt caggcattca taaggcaggc 2187 actatcagaa agtgtacgcc aactaaggga cccacaaagc aggcagaggt aatgcagaaa 2247 tctgttttgt tcccatgaaa tcaccaatca aggcctccgt tcttctaaag attagtccat 2307 catcattagc aactgagatc aaagcactct tccactttac gtgattaaaa tcaaacctgt 2367 atcagcaagt taaatggttc catttctgtg atttttctat tatttgaggg gagttggcag 2427 aagttccatg tatatgggat ctttacaggt cagatcttgt tacaggaaat ttcaaaggtt 2487 tgggagtggg gagggaaaaa agctcagtca gtgaggatca ttttatcaca ttagactggg 2547 gcagaactct gccaggattt aggaatattt tcagaacaga ttttagatat tatttctatc 2607 catatattga aaagaatacc attgtcaatc ttattttttt aaaagtactc agtgtagaaa 2667 ttgctagccc ttaattcttt tccagctttt catattaatg tatgcagagt ctcaccaagc 2727 tcaaagacac tggttggggg tggagggtgc cacagggaaa gctgtagaag gcaagaagac 2787 tcgagaatcc cccagagtta tttttctcca taaagaccat cagagtgctt aactgagctg 2847 ttggagactg tgaggcattt aggaaaaaaa tagcccactc acatcattcc ttgtaagtct 2907 taagttcatt ttcattttac gtggaggaaa aaaatttaaa aagctattag tatttattaa 2967 tgaattttac tgagacattt cttagaaata tgcacttcta tactagcaag ctctgtctct 3027 aaaatgcaag ttggcctttt gcttgccaca tttctgcatt aaacttctat attagcttca 3087 aaggctttta aactcaatgc gaacattcta cgggatgttc ttagatgcct ttaaaaaggg 3147 ggcagatcta attttatttg aaccctcact ttccaacttc accatgaccc agtactagag 3207 attagggcac ttcaaagcat tgaaaaaaat ctactgatac ttactttctt agacaagtag 3267 ttcttagtta accaccaatg gaactgggtt cattctgaat cctggaggag cttcctcgtg 3327 ccacccagtg tttctgggcc ctctgtgtga gcagccaggt atgagctgtt ttagaagcag 3387 cgtgttgcct tcatctctcc cgtttcccaa aagaacaaag gataaaggtg acagtcacac 3447 tcctgggtta aaaaaagcat tccagaacca cttctcttta tgggcacaac aaagaaacga 3507 aggctgaagt tcgcctaccc aaaatgaaaa gtaggcttta cagtcaaaag tacttctgtt 3567 gattgctaaa taacttcatt ttcttgaaat agagcaactt tgagtgaaat ctgcaacatg 3627 gataccatgt atataagata ctgctgtaca gaagagttaa ggcttacagt gcaaatgagg 3687 cgtcagcttt gggtgctaaa attaacaagt ctaatattat taccatcaat caggaagaga 3747 ataataaatg tttaaacaaa cacagcagtc tgtataaaaa taccgtgtat catttactct 3807 ttctgcagct ctatacgata ggcaggagag gcttatgtgg cagcacaagc caggtgggga 3867 ttttgtaacg aagtgataaa acatttgtaa gtaatccaag taggtgtatt aaggcaccaa 3927 aagtaacatg gcacccaaca cccaaaaata aaaatatgaa atatgagtgt gaactctgag 3987 tagagtatga aacaccacag aaagtcttag aaatagctct ggagtggctc tcccaggaca 4047 gtttccagtt gctgaatagt cttttggcac tgatgttcta cttcttcaca ttcatctaaa 4107 aaaaaaaaaa aaaaaaatca aaattaaaat ctgagtcagt ctgcctgcct cggttctcat 4167 tagtttaatt cttaatgcct tgcactttcc agcaatcatt caatcaaaag agtgaaatga 4227 agcacattaa caaagcagga ggcgccacgg accgcctccc tccacaccgc tccttccgcc 4287 ttcattcctt gcccacaggc ttgcactgga agctgaataa gaatccccaa aactcaaact 4347 tcctagggat gccacccctt tagtagctca cacctccccc ctccaagagc taagaaacaa 4407 aggagaatgt acttttgtag cttagataag caatgaatca gtaaaggact gatctacttg 4467 ctccaccacc cctcccttaa taataacatt tactgttatt tcctgggcct aagacttatg 4527 ttccagaact gtcacagctc cccatgtcac acccactagc ttgtgatctt tgtcaaataa 4587 ctgaaatctt ttaagcctct agtttcttcc tttgtaaaac agagataaaa tgttgtggtt 4647 tttaagtgag ataatccaag taaagcacct aacatggagt agtgaatgaa catcggttgc 4707 tactaaaagt ggacatccta ccgcatcctt aatgccacta ggcatttcca tacaatctgg 4767 ggaccaaaac ttcaatcata taaatgtatg aggttaatta aaaacactac tgtaatctgc 4827 ttgtatgatc acaaaccacc acaaaagaaa agatcgtgaa gattacactg taaacggact 4887 ctcaaatgat caggaggtgg tcacttcgca acttgctccc tccacccaac tcaaaacagg 4947 agctcgagcc tgcctgtatt tgagactgga gctgcctgta tgaggactgg atcaactgct 5007 agtcacgtta tatccaaatc tgcattatca ttgggcacat tttcacagaa ttttactgaa 5067 ttattcctta attgtttaat ggttgggaat agtttgggaa ttaccttcca tcaactctgc 5127 taagaaagga atggattctg gtagcaagac aatataattc tcctttagtt tttcagccag 5187 tgctaacaca gtaatcaaag cagcaaatcg aacctgaaag ggataaaaga gcaaagaaat 5247 aaaaagtagt gttactgtat ttattatctt aagagctgta ctgacttgag acaagctcta 5307 actttttaaa cattagttca cacgcgttta ttcacttcat tatgttcatt aagctttcat 5367 cttagaatac cagtttcacc atttgggagc tgtttgtaat atgtgcaacc ttataaatag 5427 tgttttccaa actgtgtccc aggactgcaa atctttaatg tgaaatgtct ttttataatc 5487 tcttccttta aaaaaaacca ataaaataaa atgccacatg caaactc 5534 4 2471 DNA Homo sapiens misc_feature 201..203 ATG 4 agccgcccac ggacgccaga gccgggaacc ctgacggcac ttagctgctg acaaacaacc 60 tgctccgtgg agcgcctgaa acaccagtct ttggggccag tgcctcagtt tcaatccagg 120 taacctttaa atgaaacttg cctaaaatct taggtcatac acagaagaga ctccaatcga 180 caagaagctg gaaaagaatg atg ttg tcc tta aac aac cta cag aat atc atc 233 Met Leu Ser Leu Asn Asn Leu Gln Asn Ile Ile 1 5 10 tat aac ccg gta atc ccg tat gtt ggc acc att ccc gat cag ctg gat 281 Tyr Asn Pro Val Ile Pro Tyr Val Gly Thr Ile Pro Asp Gln Leu Asp 15 20 25 cct gga act ttg att gtg ata tgt ggg cat gtt cct agt gac gca gac 329 Pro Gly Thr Leu Ile Val Ile Cys Gly His Val Pro Ser Asp Ala Asp 30 35 40 aga ttc cag gtg gat ctg cag aat ggc agc agt gtg aaa cct cga gcc 377 Arg Phe Gln Val Asp Leu Gln Asn Gly Ser Ser Val Lys Pro Arg Ala 45 50 55 gat gtg gcc ttt cat ttc aat cct cgt ttc aaa agg gcc ggc tgc att 425 Asp Val Ala Phe His Phe Asn Pro Arg Phe Lys Arg Ala Gly Cys Ile 60 65 70 75 gtt tgc aat act ttg ata aat gaa aaa tgg gga cgg gaa gag atc acc 473 Val Cys Asn Thr Leu Ile Asn Glu Lys Trp Gly Arg Glu Glu Ile Thr 80 85 90 tat gac acg cct ttc aaa aga gaa aag tct ttt gag atc gtg att atg 521 Tyr Asp Thr Pro Phe Lys Arg Glu Lys Ser Phe Glu Ile Val Ile Met 95 100 105 gtg cta aag gac aaa ttc cag gtg gct gta aat gga aaa cat act ctg 569 Val Leu Lys Asp Lys Phe Gln Val Ala Val Asn Gly Lys His Thr Leu 110 115 120 ctc tat ggc cac agg atc ggc cca gag aaa ata gac act ctg ggc att 617 Leu Tyr Gly His Arg Ile Gly Pro Glu Lys Ile Asp Thr Leu Gly Ile 125 130 135 tat ggc aaa gtg aat att cac tca att ggt ttt agc ttc agc tcg gac 665 Tyr Gly Lys Val Asn Ile His Ser Ile Gly Phe Ser Phe Ser Ser Asp 140 145 150 155 tta caa agt acc caa gca tct agt ctg gaa ctg aca gag ata agt aga 713 Leu Gln Ser Thr Gln Ala Ser Ser Leu Glu Leu Thr Glu Ile Ser Arg 160 165 170 gaa aat gtt cca aag tct ggc acg ccc cag ctt agc ctg cca ttc gct 761 Glu Asn Val Pro Lys Ser Gly Thr Pro Gln Leu Ser Leu Pro Phe Ala 175 180 185 gca agg ttg aac acc ccc atg ggc cct gga cga act gtc gtc gtt aaa 809 Ala Arg Leu Asn Thr Pro Met Gly Pro Gly Arg Thr Val Val Val Lys 190 195 200 gga gaa gtg aat gca aat gcc aaa agc ttt aat gtt gac cta cta gca 857 Gly Glu Val Asn Ala Asn Ala Lys Ser Phe Asn Val Asp Leu Leu Ala 205 210 215 gga aaa tca aag gat att gct cta cac ttg aac cca cgc ctg aat att 905 Gly Lys Ser Lys Asp Ile Ala Leu His Leu Asn Pro Arg Leu Asn Ile 220 225 230 235 aaa gca ttt gta aga aat tct ttt ctt cag gag tcc tgg gga gaa gaa 953 Lys Ala Phe Val Arg Asn Ser Phe Leu Gln Glu Ser Trp Gly Glu Glu 240 245 250 gag aga aat att acc tct ttc cca ttt agt cct ggg atg tac ttt gag 1001 Glu Arg Asn Ile Thr Ser Phe Pro Phe Ser Pro Gly Met Tyr Phe Glu 255 260 265 atg ata att tac tgt gat gtt aga gaa ttc aag gtt gca gta aat ggc 1049 Met Ile Ile Tyr Cys Asp Val Arg Glu Phe Lys Val Ala Val Asn Gly 270 275 280 gta cac agc ctg gag tac aaa cac aga ttt aaa gag ctc agc agt att 1097 Val His Ser Leu Glu Tyr Lys His Arg Phe Lys Glu Leu Ser Ser Ile 285 290 295 gac acg ctg gaa att aat gga gac atc cac tta ctg gaa caa tca ttc 1145 Asp Thr Leu Glu Ile Asn Gly Asp Ile His Leu Leu Glu Gln Ser Phe 300 305 310 315 aat caa aag agt gaa atg aag cac att aac aaa gca gga ggc gcc acg 1193 Asn Gln Lys Ser Glu Met Lys His Ile Asn Lys Ala Gly Gly Ala Thr 320 325 330 gac cgc ctc cct cca cac cgc tcc ttc cgc ctt cat tcc ttg ccc aca 1241 Asp Arg Leu Pro Pro His Arg Ser Phe Arg Leu His Ser Leu Pro Thr 335 340 345 ggc ttg cac tgg aag ctg aat aag aat ccc caa aac tca aac ttc cta 1289 Gly Leu His Trp Lys Leu Asn Lys Asn Pro Gln Asn Ser Asn Phe Leu 350 355 360 ggg atg cca ccc ctt tag tagctcacac ctcccccctc caagagctaa 1337 Gly Met Pro Pro Leu * 365 gaaacaaagg agaatgtact tttgtagctt agataagcaa tgaatcagta aaggactgat 1397 ctacttgctc caccacccct cccttaataa taacatttac tgttatttcc tgggcctaag 1457 acttatgttc cagaactgtc acagctcccc atgtcacacc cactagcttg tgatctttgt 1517 caaataactg aaatctttta agcctctagt ttcttccttt gtaaaacaga gataaaatgt 1577 tgtggttttt aagtgagata atccaagtaa agcacctaac atggagtagt gaatgaacat 1637 cggttgctac taaaagtgga catcctaccg catccttaat gccactaggc atttccatac 1697 aatctgggga ccaaaacttc aatcatataa atgtatgagg ttaattaaaa acactactgt 1757 aatctgcttg tatgatcaca aaccaccaca aaagaaaaga tcgtgaagat tacactgtaa 1817 acggactctc aaatgatcag gaggtggtca cttcgcaact tgctccctcc acccaactca 1877 aaacaggagc tcgagcctgc ctgtatttga gactggagct gcctgtatga ggactggatc 1937 aactgctagt cacgttatat ccaaatctgc attatcattg ggcacatttt cacagaattt 1997 tactgaatta ttccttaatt gtttaatggt tgggaatagt ttgggaatta ccttccatca 2057 actctgctaa gaaaggaatg gattctggta gcaagacaat ataattctcc tttagttttt 2117 cagccagtgc taacacagta atcaaagcag caaatcgaac ctgaaaggga taaaagagca 2177 aagaaataaa aagtagtgtt actgtattta ttatcttaag agctgtactg acttgagaca 2237 agctctaact ttttaaacat tagttcacac gcgtttattc acttcattat gttcattaag 2297 ctttcatctt agaataccag tttcaccatt tgggagctgt ttgtaatatg tgcaacctta 2357 taaatagtgt tttccaaact gtgtcccagg actgcaaatc tttaatgtga aatgtctttt 2417 tataatctct tcctttaaaa aaaaccaata aaataaaatg ccacatgcaa actc 2471 5 316 PRT Homo sapiens VARIANT 18 5-2-162 polymorphic amino acid Tyr or Phe 5 Met Leu Ser Leu Asn Asn Leu Gln Asn Ile Ile Tyr Asn Pro Val Ile 1 5 10 15 Pro Tyr Val Gly Thr Ile Pro Asp Gln Leu Asp Pro Gly Thr Leu Ile 20 25 30 Val Ile Cys Gly His Val Pro Ser Asp Ala Asp Arg Phe Gln Val Asp 35 40 45 Leu Gln Asn Gly Ser Ser Val Lys Pro Arg Ala Asp Val Ala Phe His 50 55 60 Phe Asn Pro Arg Phe Lys Arg Ala Gly Cys Ile Val Cys Asn Thr Leu 65 70 75 80 Ile Asn Glu Lys Trp Gly Arg Glu Glu Ile Thr Tyr Asp Thr Pro Phe 85 90 95 Lys Arg Glu Lys Ser Phe Glu Ile Val Ile Met Val Leu Lys Asp Lys 100 105 110 Phe Gln Val Ala Val Asn Gly Lys His Thr Leu Leu Tyr Gly His Arg 115 120 125 Ile Gly Pro Glu Lys Ile Asp Thr Leu Gly Ile Tyr Gly Lys Val Asn 130 135 140 Ile His Ser Ile Gly Phe Ser Phe Ser Ser Asp Leu Gln Ser Thr Gln 145 150 155 160 Ala Ser Ser Leu Glu Leu Thr Glu Ile Ser Arg Glu Asn Val Pro Lys 165 170 175 Ser Gly Thr Pro Gln Leu Ser Leu Pro Phe Ala Ala Arg Leu Asn Thr 180 185 190 Pro Met Gly Pro Gly Arg Thr Val Val Val Lys Gly Glu Val Asn Ala 195 200 205 Asn Ala Lys Ser Phe Asn Val Asp Leu Leu Ala Gly Lys Ser Lys Asp 210 215 220 Ile Ala Leu His Leu Asn Pro Arg Leu Asn Ile Lys Ala Phe Val Arg 225 230 235 240 Asn Ser Phe Leu Gln Glu Ser Trp Gly Glu Glu Glu Arg Asn Ile Thr 245 250 255 Ser Phe Pro Phe Ser Pro Gly Met Tyr Phe Glu Met Ile Ile Tyr Cys 260 265 270 Asp Val Arg Glu Phe Lys Val Ala Val Asn Gly Val His Ser Leu Glu 275 280 285 Tyr Lys His Arg Phe Lys Glu Leu Ser Ser Ile Asp Thr Leu Glu Ile 290 295 300 Asn Gly Asp Ile His Leu Leu Glu Val Arg Ser Trp 305 310 315 6 358 PRT Homo sapiens VARIANT 18 5-2-162 polymorphic amino acid Tyr or Phe 6 Met Leu Ser Leu Asn Asn Leu Gln Asn Ile Ile Tyr Asn Pro Val Ile 1 5 10 15 Pro Tyr Val Gly Thr Ile Pro Asp Gln Leu Asp Pro Gly Thr Leu Ile 20 25 30 Val Ile Cys Gly His Val Pro Ser Asp Ala Asp Arg Phe Gln Val Asp 35 40 45 Leu Gln Asn Gly Ser Ser Val Lys Pro Arg Ala Asp Val Ala Phe His 50 55 60 Phe Asn Pro Arg Phe Lys Arg Ala Gly Cys Ile Val Cys Asn Thr Leu 65 70 75 80 Ile Asn Glu Lys Trp Gly Arg Glu Glu Ile Thr Tyr Asp Thr Pro Phe 85 90 95 Lys Arg Glu Lys Ser Phe Glu Ile Val Ile Met Val Leu Lys Asp Lys 100 105 110 Phe Gln Val Ala Val Asn Gly Lys His Thr Leu Leu Tyr Gly His Arg 115 120 125 Ile Gly Pro Glu Lys Ile Asp Thr Leu Gly Ile Tyr Gly Lys Val Asn 130 135 140 Ile His Ser Ile Gly Phe Ser Phe Ser Ser Asp Leu Gln Ser Thr Gln 145 150 155 160 Ala Ser Ser Leu Glu Leu Thr Glu Ile Ser Arg Glu Asn Val Pro Lys 165 170 175 Ser Gly Thr Pro Gln Leu Pro Ser Asn Arg Gly Gly Asp Ile Ser Lys 180 185 190 Ile Ala Pro Arg Thr Val Tyr Thr Lys Ser Lys Asp Ser Thr Val Asn 195 200 205 His Thr Leu Thr Cys Thr Lys Ile Pro Pro Met Asn Tyr Val Ser Lys 210 215 220 Ser Leu Pro Phe Ala Ala Arg Leu Asn Thr Pro Met Gly Pro Gly Arg 225 230 235 240 Thr Val Val Val Lys Gly Glu Val Asn Ala Asn Ala Lys Ser Phe Asn 245 250 255 Val Asp Leu Leu Ala Gly Lys Ser Lys Asp Ile Ala Leu His Leu Asn 260 265 270 Pro Arg Leu Asn Ile Lys Ala Phe Val Arg Asn Ser Phe Leu Gln Glu 275 280 285 Ser Trp Gly Glu Glu Glu Arg Asn Ile Thr Ser Phe Pro Phe Ser Pro 290 295 300 Gly Met Tyr Phe Glu Met Ile Ile Tyr Cys Asp Val Arg Glu Phe Lys 305 310 315 320 Val Ala Val Asn Gly Val His Ser Leu Glu Tyr Lys His Arg Phe Lys 325 330 335 Glu Leu Ser Ser Ile Asp Thr Leu Glu Ile Asn Gly Asp Ile His Leu 340 345 350 Leu Glu Val Arg Ser Trp 355 7 368 PRT Homo sapiens VARIANT 18 5-2-162 polymorphic amino acid Tyr or Phe 7 Met Leu Ser Leu Asn Asn Leu Gln Asn Ile Ile Tyr Asn Pro Val Ile 1 5 10 15 Pro Tyr Val Gly Thr Ile Pro Asp Gln Leu Asp Pro Gly Thr Leu Ile 20 25 30 Val Ile Cys Gly His Val Pro Ser Asp Ala Asp Arg Phe Gln Val Asp 35 40 45 Leu Gln Asn Gly Ser Ser Val Lys Pro Arg Ala Asp Val Ala Phe His 50 55 60 Phe Asn Pro Arg Phe Lys Arg Ala Gly Cys Ile Val Cys Asn Thr Leu 65 70 75 80 Ile Asn Glu Lys Trp Gly Arg Glu Glu Ile Thr Tyr Asp Thr Pro Phe 85 90 95 Lys Arg Glu Lys Ser Phe Glu Ile Val Ile Met Val Leu Lys Asp Lys 100 105 110 Phe Gln Val Ala Val Asn Gly Lys His Thr Leu Leu Tyr Gly His Arg 115 120 125 Ile Gly Pro Glu Lys Ile Asp Thr Leu Gly Ile Tyr Gly Lys Val Asn 130 135 140 Ile His Ser Ile Gly Phe Ser Phe Ser Ser Asp Leu Gln Ser Thr Gln 145 150 155 160 Ala Ser Ser Leu Glu Leu Thr Glu Ile Ser Arg Glu Asn Val Pro Lys 165 170 175 Ser Gly Thr Pro Gln Leu Ser Leu Pro Phe Ala Ala Arg Leu Asn Thr 180 185 190 Pro Met Gly Pro Gly Arg Thr Val Val Val Lys Gly Glu Val Asn Ala 195 200 205 Asn Ala Lys Ser Phe Asn Val Asp Leu Leu Ala Gly Lys Ser Lys Asp 210 215 220 Ile Ala Leu His Leu Asn Pro Arg Leu Asn Ile Lys Ala Phe Val Arg 225 230 235 240 Asn Ser Phe Leu Gln Glu Ser Trp Gly Glu Glu Glu Arg Asn Ile Thr 245 250 255 Ser Phe Pro Phe Ser Pro Gly Met Tyr Phe Glu Met Ile Ile Tyr Cys 260 265 270 Asp Val Arg Glu Phe Lys Val Ala Val Asn Gly Val His Ser Leu Glu 275 280 285 Tyr Lys His Arg Phe Lys Glu Leu Ser Ser Ile Asp Thr Leu Glu Ile 290 295 300 Asn Gly Asp Ile His Leu Leu Glu Gln Ser Phe Asn Gln Lys Ser Glu 305 310 315 320 Met Lys His Ile Asn Lys Ala Gly Gly Ala Thr Asp Arg Leu Pro Pro 325 330 335 His Arg Ser Phe Arg Leu His Ser Leu Pro Thr Gly Leu His Trp Lys 340 345 350 Leu Asn Lys Asn Pro Gln Asn Ser Asn Phe Leu Gly Met Pro Pro Leu 355 360 365 8 1738 DNA Mus musculus 8 gagtgttact accaccgggg acaagttttt actttgagta atccttaaat gaagagtggg 60 taaagtgtgt atacggaaga gagactccaa tcaacaatat caataagttg aaaaagaaaa 120 atg ttg tcc tta aat aac cta caa aat atc atc tat aac ccg ata atc 168 Met Leu Ser Leu Asn Asn Leu Gln Asn Ile Ile Tyr Asn Pro Ile Ile 1 5 10 15 ccc tat gtt ggc acc att act gag caa ttg aag cct ggc tct ctg att 216 Pro Tyr Val Gly Thr Ile Thr Glu Gln Leu Lys Pro Gly Ser Leu Ile 20 25 30 gta atc cgt ggg cat gtc cct aaa gat tca gaa aga ttc cag gtt gac 264 Val Ile Arg Gly His Val Pro Lys Asp Ser Glu Arg Phe Gln Val Asp 35 40 45 ttt cag ctg ggc aac agc ctg aag cca aga gca gac gtg gcc ttc cac 312 Phe Gln Leu Gly Asn Ser Leu Lys Pro Arg Ala Asp Val Ala Phe His 50 55 60 ttt aac cct cgg ttc aaa agg tct agc tgc att gtt tgt aac aca ctg 360 Phe Asn Pro Arg Phe Lys Arg Ser Ser Cys Ile Val Cys Asn Thr Leu 65 70 75 80 aca cag gag aag tgg ggc tgg gag gag atc acc tac gac atg ccc ttc 408 Thr Gln Glu Lys Trp Gly Trp Glu Glu Ile Thr Tyr Asp Met Pro Phe 85 90 95 aga aaa gaa aag tcc ttt gag atc gtg ttc atg gtg ctc aag aac aaa 456 Arg Lys Glu Lys Ser Phe Glu Ile Val Phe Met Val Leu Lys Asn Lys 100 105 110 ttc cag gtg gct gtg aac gga agg cat gtt ctg ctg tac gcc cac agg 504 Phe Gln Val Ala Val Asn Gly Arg His Val Leu Leu Tyr Ala His Arg 115 120 125 atc agc ccg gag cag atc gac aca gtg ggc atc tac ggc aaa gtg aac 552 Ile Ser Pro Glu Gln Ile Asp Thr Val Gly Ile Tyr Gly Lys Val Asn 130 135 140 atc cac tcc atc ggg ttc aga ttc agc tcg gat tta cag agt atg gaa 600 Ile His Ser Ile Gly Phe Arg Phe Ser Ser Asp Leu Gln Ser Met Glu 145 150 155 160 aca tct gct ctg gga ctg aca cag ata aac aga gag aat ata caa aag 648 Thr Ser Ala Leu Gly Leu Thr Gln Ile Asn Arg Glu Asn Ile Gln Lys 165 170 175 cca ggc aag ctc cag ctg agc ctg cca ttt gaa gca agg ttg aat gcc 696 Pro Gly Lys Leu Gln Leu Ser Leu Pro Phe Glu Ala Arg Leu Asn Ala 180 185 190 tcc atg ggt cct gga cga acc gtt gtc att aaa ggg gaa gtg aac acc 744 Ser Met Gly Pro Gly Arg Thr Val Val Ile Lys Gly Glu Val Asn Thr 195 200 205 aat gcc cga agc ttt aat gtt gac cta gtg gca gga aaa aca agg gat 792 Asn Ala Arg Ser Phe Asn Val Asp Leu Val Ala Gly Lys Thr Arg Asp 210 215 220 atc gct ctg cac ttg aac cca cgc ctc aat gtg aaa gca ttt gta aga 840 Ile Ala Leu His Leu Asn Pro Arg Leu Asn Val Lys Ala Phe Val Arg 225 230 235 240 aat tcc ttt ctt cag gat gcc tgg gga gaa gag gag aga aat att acc 888 Asn Ser Phe Leu Gln Asp Ala Trp Gly Glu Glu Glu Arg Asn Ile Thr 245 250 255 tgc ttc cca ttt agt tct ggg atg tac ttt gag atg ata atc tac tgt 936 Cys Phe Pro Phe Ser Ser Gly Met Tyr Phe Glu Met Ile Ile Tyr Cys 260 265 270 gat gtc cgg gaa ttc aag gtt gct ata aat ggt gtg cac agc ctg gag 984 Asp Val Arg Glu Phe Lys Val Ala Ile Asn Gly Val His Ser Leu Glu 275 280 285 tac aaa cac aga ttt aaa gac cta agc agt att gat aca cta tca gtc 1032 Tyr Lys His Arg Phe Lys Asp Leu Ser Ser Ile Asp Thr Leu Ser Val 290 295 300 gat ggt gat atc cgt ttg ctg gat gta agg agc tgg tag ctaccatgac 1081 Asp Gly Asp Ile Arg Leu Leu Asp Val Arg Ser Trp * 305 310 315 tgccaaaacc cccgaaatac aaaatggctt atccggtact ggccatgtca aatgcatctc 1141 gctttcacca tattgtttat attgctaagt tgagctcctc caacatcaag tcctactggt 1201 gttgtcaggt ctggccatgc agtacattca gaggaacaga gccggggcaa tcacagctca 1261 ctgccagaga ggctctgcac actgggtccc tcttataaac cacactcagc aaatatttaa 1321 gtgcctaata tactacatat actagctaat agggatggca agcatacttc ctttgtatat 1381 tctctgagcc gggcacagac atggcagggc ccagaacttg tgtggtccat gttttctagc 1441 acttcgtacc agtttctggc ctcctaatgt agggtcttct tgctggcatt gcattaaccc 1501 cactaggggc ctttgcagtt aaggtcagaa aaatatacta atggatggca aacactactt 1561 ccccagcaac ccttttcata atcagcattc tatcatatct cataattgaa gactgcatag 1621 catttactta gctctcaccg ctttaaactt tataaaatgt atgatgctga acacagcaga 1681 aaaactgagg ccaaaaccct gaattatgac aaaacaagtg ttctgctcca agcagat 1738 9 316 PRT Mus musculus 9 Met Leu Ser Leu Asn Asn Leu Gln Asn Ile Ile Tyr Asn Pro Ile Ile 1 5 10 15 Pro Tyr Val Gly Thr Ile Thr Glu Gln Leu Lys Pro Gly Ser Leu Ile 20 25 30 Val Ile Arg Gly His Val Pro Lys Asp Ser Glu Arg Phe Gln Val Asp 35 40 45 Phe Gln Leu Gly Asn Ser Leu Lys Pro Arg Ala Asp Val Ala Phe His 50 55 60 Phe Asn Pro Arg Phe Lys Arg Ser Ser Cys Ile Val Cys Asn Thr Leu 65 70 75 80 Thr Gln Glu Lys Trp Gly Trp Glu Glu Ile Thr Tyr Asp Met Pro Phe 85 90 95 Arg Lys Glu Lys Ser Phe Glu Ile Val Phe Met Val Leu Lys Asn Lys 100 105 110 Phe Gln Val Ala Val Asn Gly Arg His Val Leu Leu Tyr Ala His Arg 115 120 125 Ile Ser Pro Glu Gln Ile Asp Thr Val Gly Ile Tyr Gly Lys Val Asn 130 135 140 Ile His Ser Ile Gly Phe Arg Phe Ser Ser Asp Leu Gln Ser Met Glu 145 150 155 160 Thr Ser Ala Leu Gly Leu Thr Gln Ile Asn Arg Glu Asn Ile Gln Lys 165 170 175 Pro Gly Lys Leu Gln Leu Ser Leu Pro Phe Glu Ala Arg Leu Asn Ala 180 185 190 Ser Met Gly Pro Gly Arg Thr Val Val Ile Lys Gly Glu Val Asn Thr 195 200 205 Asn Ala Arg Ser Phe Asn Val Asp Leu Val Ala Gly Lys Thr Arg Asp 210 215 220 Ile Ala Leu His Leu Asn Pro Arg Leu Asn Val Lys Ala Phe Val Arg 225 230 235 240 Asn Ser Phe Leu Gln Asp Ala Trp Gly Glu Glu Glu Arg Asn Ile Thr 245 250 255 Cys Phe Pro Phe Ser Ser Gly Met Tyr Phe Glu Met Ile Ile Tyr Cys 260 265 270 Asp Val Arg Glu Phe Lys Val Ala Ile Asn Gly Val His Ser Leu Glu 275 280 285 Tyr Lys His Arg Phe Lys Asp Leu Ser Ser Ile Asp Thr Leu Ser Val 290 295 300 Asp Gly Asp Ile Arg Leu Leu Asp Val Arg Ser Trp 305 310 315 10 18 DNA Artificial Sequence sequencing oligonucleotide PrimerPU 10 tgtaaaacga cggccagt 18 11 18 DNA Artificial Sequence sequencing oligonucleotide PrimerRP 11 caggaaacag ctatgacc 18 12 106746 DNA Homo sapiens misc_feature 1..68647 5′regulation region 12 ttggcttggc agggcaacca gctcaccaga ctctctgcag acccgaagtc attacataca 60 gtatgataac agggaatgga cccgaccagc atttgctgga gatgatatct ggtgtcagcc 120 cgacaggccc ctacctgctt ctcttgatat gcaggaatcc cttcaagctc caacaagatc 180 tgtttaatag actggagagt cctttagttc cttcctctaa gggaaaatca gatcgttctg 240 gtttgcttgg taactcctta cttcatccct gatgggaagt ttatagaatg aggaaccagg 300 gctattacat gaaactataa aactgcctag agcacatact tggtattttt aacattgttg 360 agagggactc acttaattca gccttgcagc tattgcattc cwgtccaaac caacggcagg 420 ttctcaaaac aagcggtgaa agggttcctg ttgcagagct gtctggacat ttaaagaagg 480 gagaggaaat ctcaaggggt cggttgcact ggaatagaaa tcgcctgttc tttttttttg 540 agacggagtc tcgctctgtc acccaggctg gagagcagtt gcgcgatctt tgctcactgc 600 aacctctgcc tcccgggttc acgccattct cctgcctcag cctcctgaat agctgggact 660 acaggcgccc gccaccacgt ctggctcatt ttttgtattt ttagtagaga tggagtttca 720 ccattttagc caggatggtc tcgatctgct gaccttgtaa tccacccgcc tcggcctccc 780 aaagtacagg gattataggc gtgagccacc gcgcccaggt gcctgttcct tttttaagag 840 tctcactctg tcgcccaggc tggcgtgcag tggcgcgatc tctgcttact gcagtctccg 900 tctcctgagt tcaaatcaag cgagaaatca cttgttctct tctgtgaacg gaagcatcgc 960 agatctctct tggcctcaca ctcctccatc tccctgattc ctctgttctt catttaccta 1020 ccttcccagc agtctgcaga gctggccgct cactcacctc tagtaagggg atggagggtc 1080 ctgtgttgga ataactcact gaccgctaga aagttaaaaa taaatgggta atgccaggag 1140 aacttggctg gtgccttaaa agccatagaa cttctctttc catctgtaga taactgtaga 1200 caattttgtc caaaacagat aatgatctga ttctacctcc cattggtatt tcccttcctc 1260 ggcctgtgac atctcacttt ctctagactg aactttatcc cagactgtga ccttgccatg 1320 accttcctcc tgcgtgtgcc tctgccacca caggaatggc cacgcctcag atcatgtcac 1380 cgctgggaac aaaccctcta cctgcgactc tgaagttccc tctctgaccc tcttttcttt 1440 cttcccctcc cccctcccct cactccctct gcacctgtgt ttcgctgtca cgctcccaac 1500 tcatccctgt agagctggtg aagagatgct gatgtagttc ttgaccttga accccagccc 1560 tgcagccgtc ctgtggcctc actgacccag cgtcatgccc tggtcaagca ttttggtgat 1620 gctcttggtg attttcaatg ggacctgcct tgccaagccc tgggcttagg tgaaccagga 1680 ccacctgcat tctatgtttt tgattgctgg aaaaaaatca tgaaatgtca actgttgttc 1740 tcatttttcc cactgccagt tcctgctacc caacctccgc cctcatttca aggccttgag 1800 tacttttttt ctatagtgaa gtctcccaaa aatgatattt ttttaaaaaa gaaaagccat 1860 agtactctga tttgatgtgg tctgttaata cctatgggct ttgacttgtt tctgctttta 1920 gacctagaca aaataaaata tctgtggtaa aacatattca agtttaccgg gcacgggggc 1980 tcacgcctgt aatcccagca ctttgggagg ctggggcagg cagatcactt gagcccagga 2040 gtttgagacc agcctgggca acagggtgaa acaacatctc tacaaaaata caaaaaatac 2100 ctgggcattg tggtgcatgc ctgtagtcgc agctactcgg gagactgagg tgggaggatg 2160 gcttgagctc tggaggcgga ggtcatagtg agccaagatc gtgccactga actccagcct 2220 gggcaacaga ggcagattct ttctctctaa aaaacataaa ataaaaaaag gccaggcgca 2280 gtggctcaca cctgtaatcc cagcactttg ggaggctgag gggggcggac gaagaggtca 2340 ggagatagag accatcctgg ccaacatggt gaaaccctgc ctctactaaa aatacaaaaa 2400 ttagccgggt gtagtggtgc atgcctgtaa tctcaactac tcaggaggct gaggcaggaa 2460 aatcgcttga acccaggagg cggaggctgc agtgagccaa gatcgcacca ctgcactcca 2520 gcctgggtga cagagcaaga ctctgtcccc caccaaaaaa aataaataaa taaatcaggc 2580 caaagggcaa aaatgcttgc tttttagcac ttagtagtta tttccccaag aagagcggga 2640 gagaagttta ttaataatga aactggacag ttctttatca gctctaattg tttgactcaa 2700 tggcttctct tctcattacc atgcagtgct ctgctggctg caatgccttt gaacttcaca 2760 agaaggttag aatttcactg agacattcgg atggtgtggg tgtcagggtg cagctctcac 2820 acatagttga gagtgtaaat tgatacaact ttatggaaaa ttaattggga gtacccattc 2880 acactcctgt ctagcaatct cactttaagg acttgatcct acagaactca ttacatggtg 2940 caaggttcac agtgtggcat tcaaaataga gaagagctgc gggtaactcc catgcccgtt 3000 ggcaggaact ggttgaataa attatggtgc atcagtgctg tggggtatca ttaaaccatt 3060 aaaaagaaga gagagtcctg gccttaaaaa aaacttatct gatgtattgt taaacagata 3120 aagcaagttg tagatcaatg tgatttgggg ctaaaaaaat atttctatat aggtgtgaac 3180 atggccatga ctaaggaatc aggaaggaag tacctagatt gtaaccagta acatgtcggg 3240 agtgagatgg gattgagaga cgtaataata gattgagaga aaaagatttt cccatctctt 3300 tttgattttt taagaaaaca gcatgatttt cagtaatttt tacttttgtg tgtttttggt 3360 attttttctt tttctttttt tttttttttt ttttgagacg gagtttcact cttgttgccc 3420 aggctggaat gcgatggccc agtcccagct cactgcaacc ttcacttccc aggttcaaga 3480 gattctcctg tctcagcctc ccgagtagct gggattacag gcccctgcca ttacgcccag 3540 ctactttttg tatttttagt agagatgggt ttcaccgtgt tggttaggct ggtttgaact 3600 cctgagctca ggcgatctgc ctacttcagc ctcccaaaat gctgggatta cagccgtgag 3660 ccaccgcccc cagccggtat tttttcaaat caaagaaaaa ataatagagt aaatcatcca 3720 aaactttaga tggtatttag actcagtaaa cttttcatat atgacagatg aagccaaatg 3780 gtctttctgt gcagtcagct agcacacaat tgtgcacccg aggaaaatta gagactgaac 3840 cggggtgtct gtggatgcat ttcctcagca ttcagccttc cttttgcccg tgttctagca 3900 ttacttctgt cctacagcct gggatttgtg aatgaaatag acaggtgcaa aaactccctg 3960 cctgtctgta atatccatag ccccgtgctc tacttgtatt tgcatgtaca aaccataatc 4020 tcctgtaaaa tactctgtga tatttctgaa taataataaa ctctacatcc tacacaaagg 4080 caaaacccct gtatctttca tctttgaaac catagcaaag gtatgaaatt acacctgagc 4140 atgcctggcc tcaaagtcct ggaacggtta tgtctttgac cctcacttca actcaactcc 4200 agaagaagca ggtcttcctt gtaattggat agaaaactca ttgtagagaa gaaagatcta 4260 caggtcaaga aacccacagg tttgctgtaa tccgagcaaa gcactgtagc atttatttta 4320 tattttcact cttcttattt agctcttttt tttttttttt ttttgagatg gagtttcatt 4380 cttgtcaccc agcctggagc aatggtgcca tctcggctca ctgcaacctc tgcctcccag 4440 gttcaagtga ttctcctgcc tcagcctcct gagtagctgg ggttacaggc tcccaccgcc 4500 acacccagcc aattttttgt atttttagta gagacggggt ttcaccatgt tagccagact 4560 ggtctcaaac tcctggcctt aggtgatcca cccgcctcag catcccaaag tgctgggatt 4620 acaggcgcac cggccttagc tcttttatcc ttaatgaaat gctcctcatt ccctgaggtc 4680 tcacttgaat tcttgcccac ctctgggttg ccttcctctt ctgtctgtgc tttgtaacac 4740 gtggttcctt atgatgtcaa tatttatgca tatgtcttca ttccattact ggattataat 4800 cttgaagcaa cagatttttg tctctatatc ccagagccta gaatggattc ttacactggg 4860 cagtaagtac ttaataaatg tatcccaaat caaataaata catttcttct ttttcttttc 4920 tttttttttt ttttttgaga cagggttcca ctctgtcacc caggctggag tgtaatgaca 4980 tgatctcagc ttactacagc ctcaatctcc tgggcttaag caatcctccc acctcagcct 5040 cccacatagc taggactaca ggcgctcacc acaacacctc atttttgtat attttttgta 5100 gagattgggg gatctcacta cgttgccccg gctggttttg aacttctggg ctcagacaat 5160 ccacccacct tggcctccca aactgttgag attacaggaa tgagccacca ttccctggcc 5220 aaatacattt ctaaaagcca gtttctggag tatactgtca aataatagat atatgtccac 5280 atttttatac ggacttatat tgtaagaaaa agtaaaaata agtgtgaagt tattacagta 5340 atagtaatta ttttgcagaa aaagaactga gtttaaacag gctttttaga aaaacccaac 5400 aggagattca cagtctggta ctaacgttta gacatggatc atcagtaaat gtgttccaaa 5460 gagttacaca gataccagct ttgtcttggg aattcttacc cctgaaaatt gattgactat 5520 cactgactgt gtgacatgag aaagttttgt ggggtttttt ttttgtattt ttttgagacg 5580 tatcttgctc tgtcacccaa gctggagtcc actggcgcga tcttggctca ctacaacctc 5640 tgccgcctgg ttcaagcgat tctcctgcct cagcctccag aatagctgcg attacaggca 5700 cctgccacca tgcccggcta atttttgtat ttttagtaaa gacggggttt catcgtgttg 5760 gccaggatgg tcttgaactc ctgacctcag gtgatctgcc cacctcagcc tcccaaagtg 5820 ctgggattac aggcatgagc caccgtgccc agcctgaaaa agttttgaac ggtctaaatc 5880 catatgctgt gaatcctatt accatcacac acttaggcat ttaaaatcat attttcaagg 5940 ccaggtactg aaatattttc tgcaagcaga gagatcaaac tttagcattg ttattcttgt 6000 agtagtttca tagtttgagg tcttagattt aagtcttcca ttgattttga tttgattttt 6060 gtatatggag ataggggtct agtttcattc ttttgcatat ggatatccag ttttcccagc 6120 accatttatt gaagagactg tctttttcac cagtgtatgc tcttggcacc ttcgtcaaaa 6180 atgagttccc tgtaggtgtg tgggtttgct tctggttctc tattctgtcc cattggtcta 6240 agtgtttggt tttatgctac taccatgctg gttggtatag ctctgcagta taacttgaaa 6300 gcaggtaatg tgattccaaa gaagctagtt aagtaattga gctaaactgg aacctcaggt 6360 gtagaagtca taagcgtggg gagcgtttct tctcaggttc tctgcctata atttagtttg 6420 ccacaccaga tgaacagtga caacttggtc ttggtgttcg tggtggtttc caaccaaact 6480 ttggtcataa caggtgaacc agcctggggc atgctttccc attcggttat cctccccata 6540 gtttgcaaag tagcaaagat gaactcttca tgagttggct aagcatagac atttcaagac 6600 caaactaaac gtcctgaaga gcatgtttca cagaaaacta gcccctaagg gaccagtggg 6660 ggctgtcaga gaacaaggtt tcaacgtact gagttttaaa gatctaattg gcttttaata 6720 acaattcatg aaccaggcac catagtctac aaaatagaca gggtttctgc tgggcactgc 6780 aggacagttg gtttttggaa ggtggcttga gcaggaacaa ggaaaaagca ccgtgccaag 6840 agtggattgg ttaacatcag gggacttcgg gtgactttcc ttctatgggt taaagcaaag 6900 gggacttccc tagcatgtca gctcaggttg actgggcccc tttggattgg ttgctgtgaa 6960 tctcctagtt ttttttgttt tggtttggtt tgggtctttt ggggaaaacg ggccagtttg 7020 gagattcagc tattatttct ctctcctgat atcagaagat cagatcttat gagtacacag 7080 ctgaggtttt gggttggtga tgtggaaccc tggtgtgagt gactccattt tgggttggtc 7140 tattggggtc tcggtgcagg agctcagtcc aaatcagtgg cctctcctca tttttatttg 7200 acttctccat caatctatcc gtgtctcccg tcacatcagt ccattccccc gtgggctgca 7260 cattcagctc ggagctgaga gcttttccca gggtgtgccc tggggtttct gctgcttgca 7320 gcctgatatt aaatctcagg tgtaaatctt cagaggcaac tgttccttag tacccagagc 7380 tttcagctcc ctgagcagaa atgggacttg actgtcagtt tataaactaa ccaaggtgtg 7440 aaattcatgc aacttagccg actttctgtt caaagaattc ttggcagcag ttaatacatt 7500 ttgcccaaat ataagataat tcccttgtac tcacaatgag aaagttttac aaaatggggg 7560 ttttctttag tttacttgaa tataaaacat aggtgttcca ctctgcagta ccttaacagt 7620 tcttaaggag atgtttgaaa caacccatgt ccaggcctca cacctcgcca attaaataaa 7680 tgagaagttc ttcccagcca gtgttaagaa aaattaacat caagttttag gaaggtagac 7740 agattatgca aatgcatacc tatatgattt aagttattac attaatttac acacacatat 7800 ttaaaatcat agattaatct aatttagaga tgctgcattt tttccatctc tcctgtttca 7860 taaatgttat tcacacggca tttctctgct atcctcggaa tagtgtttgt atcgtgtcac 7920 tctggcacgg ggctctacag aacatgtcga gcgtgttgcc ttccctactg cccacatcgt 7980 ttgagagaac acattttaaa cattttttta ttgtggtaaa atacacataa cataaaagtt 8040 acgattttaa ccttttttaa ctctgtcatc caggctggag tgcagtggcg agatcttggt 8100 tcactgcaac ctccgcctcc taggtccaag tgattctcct gcctcagcct tccgagtagc 8160 tgggattaca ggtgcacacc accacgnccg gctaattttg tatttttagt agatgcgggg 8220 tttcaccatg ttagccaggt tggtctcgaa ctcccgacct caggtgatca gcccgcctcg 8280 cctccccagt gctgggatta caggcgtgcg ccactgtgcc gggcccattt taaccacttt 8340 taagtgcaca gttcagtggc attaagtata ttcgcggtgt tgtgcgaccg tcaccaccat 8400 tcacctccag aacttctctg tcttcccaaa ctgaaattct gtacccattg aacggtaact 8460 ccccattccc catttctgct tcctaggccc tgacatggag gctgggccaa cggatatctc 8520 acctcccttc aggcttctcc agatttgccc ccgtttttct ccctctttgt cccatctcca 8580 aagaaatggt gtcttttcat catcaaggtc catcccttgc tccttgaata cactccaggc 8640 ccagtggaac aggcatcctg tggggtgcac ggacagggtg cctggggaac acccagggca 8700 cagaacccag accgggggtt tggagaaggt gtcctagcag aagtgatgtc taagctgagg 8760 ccctacagat aagagaaagt aagcagatga aagggctggg gagggtggca tttcaggcct 8820 acacaaccac acgcgtgttc ttcagccatc tccatggcct cactgcccac ctggtatcag 8880 ccggccacca cccggctaga acggctttca aaatcgctgc tcgtctactc ctcaccaaat 8940 cttgtcttca cttggtgctc aagcccatca cctttctgca agtattattt tttttttttt 9000 ggagatggag tctcgctctg tcacccgggc tggagtgcag tggttcaatg atagctcact 9060 gcaaccttga actcctgggc tcaagatcct cttgccacag cctcccaaag tgctgagatt 9120 acaggcacaa gccaccatgc gtggtccttg ctgcaacttt tttttttttt tttttttttt 9180 ttttgagaca gaatctcgct ctgtcgctca ggctggagtg cagtggtgtg atctcggctc 9240 aatgcaacct ccgcctcccg ggttcaggtg attctcctgc ctcaccctcc tgagtagcta 9300 ggaacacagg cgctcaccac cacatccagc taatttttgt gtttttagta gagccggggt 9360 tttgccatgt tggccaggct tctctcaaac tcctggacct cgggcgattg gcccgcctcg 9420 gcctcccaaa atgctggaat tacaggcatg agccaccgtg cctggccatt tgctgcaact 9480 tttgacactg ctccccctgc ttttcttccc ctctctgacc tcctttctct gctgtccttt 9540 cgttccttcc tctgccactg aagtgtcctt ctcaggtcct tctcaaggtt gtgaccttac 9600 agctgtctct tcacttccag tcatttcttt cataatcact ttgacatcct tattttcatc 9660 tcctgccctg gcctctccca gggaccagga ccatgcattc agctcctggg ggcatctcaa 9720 gcttgttgtg tgtgagcctg cccttgttgt cttctccgtc acctcttcac agcttgctct 9780 gcatttcacc tcctttcctg ttttccccag tgatcgcatc tctacagcgg ctctcacttc 9840 atccccttct ctcctagagg agtgatgcgg agtctcatta atccttgctt atgtcattct 9900 tcccccttct ctgtccatca cctccacatg tcctgttccc ccatgcgtcc tacactgtag 9960 ccaggtgggt atttcctgtg ctggtcttag acaccccctg aggataccct gcttcaggcg 10020 agagccctca gtgactccct gttgtccgga atgacgtcca gctccttgga cagtccccag 10080 tgtattcacc tgtctcatct ccttcttttc gttttgtttg tttttcttaa cttccagccc 10140 gatttctgaa tcatctccct cttgcccctc ccattgcctt tgcttaagac taaatgctcc 10200 ttcctcccaa gtccccactg cccagatttc agcagggtcc atctcaaaca tgtctgtctc 10260 caagaaactg cctctgattt ttttcataag aagacacctg tcctctctga cttcatctgt 10320 acccctctct tggaagtcac tatcttgtgc cttgcatttt cgttgtttaa gtggtctcca 10380 tttcccagca tatcttgagg tcaagggttc aggtcatttt atctttgtct atgcattgca 10440 atatgggggt ttttacatat tagctgctca ataaatcggt gttgaataaa ggcatgtgta 10500 tgctttcatt aagactatga aacccacaaa aatcagtggt tttcctattt cacccttaga 10560 aaacaaaccc acaacatagc acaacctgat attcagagct aagaacaaag gtcatgcata 10620 ttaatctaaa ttctatcttt atcaactttc acaagtaatt cgtatttccc tgtctgcatc 10680 acggggatga ttctggccag acattgacct tggtaaaatt tcctccagat tatgagaaat 10740 caagtcaaat atgccaagta acatagtttc tacttagagt caggttcatg ttttagcagg 10800 aacctcaaat accacaaaat ctgtcaagtt ctaacatttg tatctctcga cagtacctga 10860 agttcctgtt tctgtttcct cagcccaggt ttccaattca gtgagcagaa cggtgactgt 10920 gttggtaaaa gagcccacat acctgcccga tcctgcagga gtgttgcaga tgcaaacagg 10980 cgggtctcca catgacctgc ggagtaatga ctagtgtccc taaagtcatg gggcttctgg 11040 ggttagcctt gaaaaaagct aaaggttgca tagagagaga tttctatccg ttcagagact 11100 cactataatt ctctctttct gtctctgtcc ttcatctgtt tctctctttc tctctcactc 11160 tctctctctg atacacacac acacacacac acacacacac actcacactc acacactcct 11220 gagtaaggga aatgtgagaa gaaggtaaaa cttcaactaa atgaaaagaa attgtatgaa 11280 ttatggtaag caggttggtt tttagttcca gtaaagatag aaatatttag attacttagg 11340 agaaaagtct agctggtaac acatgggaat gtgcctgtgt gaaaacaaaa caaaacaaaa 11400 aatctaggct tgtggttagg tgaaggtatg tacactgctg agacatggcg atgggtgagc 11460 ttgggatgag gagaaaggct tctctgagaa gattaagaga gaaagattgt ttaaaaatgt 11520 ttaaacatgc tgggcactgt ggctcacacc tgtaatccca acactttggg aggccaaggt 11580 gggcggatca tgaggtcagg agttcgagac catcccggcc aacatggtga aaccctgtct 11640 ctgctaaaaa tacaaaaatt agccaggcgt ggtggcgggt ggctgtagtc ccagctactt 11700 gggaggctga ggcaggagaa tggcgtgaac ccaggaggcg gagatgcagt gagccgagat 11760 tgtgccactg cactccagcc tgggcgacag agcaagactc cgtctcaaaa aaaacaaaaa 11820 aacaaaaaaa aaacacacat tgacaccagg acggagttag cacatcttta caggtgagac 11880 tctcagaccc gagaaaatag aggcacttta gagctgagct aatcccacag ccacctcaac 11940 acacaaacgg ggaatctgag acccgcattg gcaccgtgcc tgaggttcta aagcccaggg 12000 cttctgactc gcctcttgtg cttcttcagt actgtgggtg ggggtggggt ggggggtgac 12060 attagctgat gagaaagatt ttggttttag aaagatggag ttaacataaa cgaaggtgta 12120 ctgggactgg tctcctctgc tgacttcatg ggaagcacac acacgcacac acacacacac 12180 acacacacac acacacacac atacacacac ctgtccaaga tcagaaaaaa tccctcacat 12240 ccctgtagca tgatcctgat tgtaaaaatg gagccctaat cagaagggca gaagcatgat 12300 tgcctctcaa gagatttgga cgccactttt tcatagttgg ttttagctgc tttgcgatat 12360 atactgaaat aaatagaaaa gggaaagaat tgtaacctgg attgacagac aacaagccct 12420 gacagacaaa aagcagataa gaaataaaat aaggaagata acccataatg taaaataaaa 12480 atagcacatt gttgcatgca ttgataccct tttttttttt tctttgagat cttgctctgt 12540 ctttcaggcc gaagtacagt gtctcaatca tagctcactg cagcctccag cttctgggct 12600 caagcaatct tcccatctca gccacccaag tagctggggc tgcaggcacg aactatggtg 12660 cccagctgat aatttttaaa aatagggaca ttagtgcatt tagcaaattt gagtgtctgc 12720 tgtgtatcaa gcactgttct gggcactggg acagcacagg gagcaaataa acaaaagccc 12780 ctgcgctcaa ggtgctcgta ttctagaggg agatgctgag ttcacctccc attaaaatgc 12840 cattctcaag atccagtccc tccacccacc ccagccccca gggttttggt ggaaatttaa 12900 ctaagttgga agattgataa tatctccatt cacatttgga tatgatttta atgaaggttg 12960 ctttttggtt tttagggaga agaaaatggc tttccagata gcactggaga tcctcttcca 13020 ggtaaatgat tgattctaaa gctatctggg ctaatagcta gtgtggctga ataaaagata 13080 atttgaggcc agggtcggtg actcatgcct gtaattccag cactttggga ggccaaggtg 13140 ggcggatcac ctgaggtcag gagttcaaga ccagcctggc caacatggta aaaccccgtc 13200 tctaccaaaa atacaaaaat tagctggttg tggtgggcgc ctgtaatccc agctactcgg 13260 aggctgaggc aggagaatcg cttgaacccg ggaggcggag gttgcagtga gccaagatca 13320 caccactgca ctccagcctg gacaacagag cgaaactcca tctcaaaaaa ttaaattaaa 13380 taaaataaat aatttgagac tatgtttatc attaacttta aaatctgtac tgcagaatag 13440 agcaactttc tacctgcggt gcactgcagg gaaagccgta tcttacaaga cttcacaaaa 13500 gccttcaaag agtattttct ctgcactaac cttcctttgc atgtgagggg cacggcaggg 13560 ttctgaatgg ggcaggttta ggatcaggcc agtcgggact gagtggattc ttcttccctc 13620 tgagttctaa gagccatagc attggtggag aacatgctgt ttgttgcttg gtggaaggga 13680 ccagaagcca gctgggtcat ctctctgttt gtgccttggc cacttaggta gccaaaggag 13740 ccctcctgac attaggtcag gtgttagtcc ctctcctttt ctgcttttag tgtgtttaag 13800 caaataaaca ttaaagttca tttctccccg ctcccctttt ttaatcataa gacagacatg 13860 tttgcaatgt ttaaatttct cattaatcag aagggatagg gagtgaggga gtaagcatta 13920 aaataagcta gcaaatggcc aggtgtggtg gctcacacct gtaatcccag gactttggga 13980 ggccaaggtg ggcagatcac ttgaggccag gagttcaaga ccagcatggc caacatggca 14040 aaactccatc tctactaaaa atacaaaaat tagccaggcg tggtgatggg cacctataat 14100 ctgagctact cgggaggctg aggcagagaa ttgcttgaac ccgggaggca aagattgcag 14160 tgagctgaga ctgcaccact gcattccagc ctgggtgaca gagcaagact ccatctcaaa 14220 aaaatgctag caaaataata ataataataa taataaaaca tacctcacca acattttcta 14280 catcttgtaa agcatacatt gactgactga agtcaccaga gttttgtttc tttctttctt 14340 aagcagggtg gggaacccgt agagccctca ggggcagcta tcatcagccc aggtaaccaa 14400 gctgaaaaac cagaaggtgc agtgcgtact caactttttc cccttagaaa cacgatatta 14460 gaaaatacac caataccaac atgtgagcaa cagttctctc tggaaggtgc agttctgggt 14520 gatttttttt tcattccata gatttttttt ttcttgagac ggagtttcgc actcttgttg 14580 cctaggctgg agtgcaatgg tgcgccacca cgcccggcta atttttgtat ttttagtaga 14640 gacggggttt caccatgttg gccaggctgg tctcgaactc ctgacctcag gtgatccacc 14700 tgcttcggcc tcctaaagtg ctgggatgac aggtgtctca ctatgttgcc taagcttttc 14760 tcgaacccct gagctcaagc ctcctcccac ctcagccatc caaagtgctg ggattacagg 14820 catgagccac cacgcctggt gagtttttat tttctttcca ctatccntat atttctaaaa 14880 tttctaacat gagctggtat cagaactgcc cctccgcatt taatctgtgt atacaaatgt 14940 atatataaca aatgatcaca tgttggtaan gtataccttg ctgcatggtg aaataaccaa 15000 ggaaacttct aaaaggttaa ctgtggttgg cctgggtaat gggagcatta attttttcca 15060 tatgctcatc tgaattttca gatttgctat gacaagcaca tatttatttt ctaattttaa 15120 aaatctatat ttaaactctt taaagactaa caccctacac actaatgtgg cacgttagct 15180 aaaataaaaa taaatacaga aatttgttta gaaatatttg taaacccttc aaggactctt 15240 ctgaatgata gtcattatta attagcaggt taattttaat caggcttctg gtcatcttca 15300 aacatttttt acttgtgtca aaatgaacca ccagagtgtg ggtttttttg ttattttttt 15360 tgtttttttg agacagagtt tcactcttgt tgcccaggct ggagtgcaat ggcgagatct 15420 cggctcactg caacctctgc ctcctgggtt caagcagctc tcctgcctca gcctcctcct 15480 gagtagctgg gattacaggc gcccaccacc acacccagct aatttttgta tttttagtag 15540 agatgggttt tgccatgttg gccaggttgg tcttgaactc ctcacctcag acgatccacc 15600 cacctcagcc tcccaaagtg ctgggactac agatgcacac caccacaccc ggttaatttt 15660 tgtattttta gtaaggacgg gggttcccca tgttggccag gctggtctca aactcctgac 15720 ctcaagtgat tcacctgcct tggcctccca aagtgctggc attacaggcc tccgccaccg 15780 cacccagccc aacctgggtc cttttgtatg tgagagtttg cttgtttttt tcacgtgctt 15840 tctctactcc agttttattc tatgacaaaa ttgaggccca acatgattta cttgcctgga 15900 tccacccaac ctgtcagtta cttcccagtg ctgctgccaa cttaatgtct ccttaaaagg 15960 atgctttaga gaaaacgaaa tcatgttgtt tttccccttt ggttaagaga tcaaacgccc 16020 accaaaagcc cttgggtcag tttcttagta gataaaaata attcttcgtc actttctgaa 16080 agcggctaac atataaccct tatgatgaat aatgtggtgt gtgtgtgtgt gcgcgcccca 16140 aattccaatg agttatcaaa gccagaaact tatattttaa atatgtttat ttcccaacca 16200 cactggaaac cacacacaga aaaaaaaaaa agcatgatta taccccctta ataaccgtta 16260 ctgcagaagg atgtgactct ccttcaacac ttgttggtat tttacagcct ccaaatctga 16320 ccatgtataa ccacctggga tagagttatt ttatttcaga accataatac ttagctatct 16380 cggaagttgc caatataaaa tgtttactct ctaatggttt tgaactaact caagacctgg 16440 ttatcccggg gagcatcctt acaaatgatc tgagagctaa cagtcctctt gcagcagtgg 16500 agggaaacac tcccgtggca atcactctcc aaaagccaga atgtgcaaga taaaagggca 16560 ccttccctgc agggaggcac attaagtcag tctgtgatct gctgccaaca tcctgactgg 16620 agccgtttct acgcctaact aatcatgacg tttgtgaatt gtgaagcttg ttgcaattca 16680 caattaactg ttaattgacc catattttat aacccgccag ccatgaactt acaagttaga 16740 tacagacact accagacatt cactattttt ttttacaatt gttttaaatg acattaatga 16800 gcatgcttga ttcctgaact cttctttaca gtataatttt aaaatatttg agtgggatac 16860 gatggagagg agggaggtgg gggaagaaat gccccatgga aaacccactc atcaggttga 16920 gagtgtggag aagccctgtg tatctgagaa ctcttaatca tccacagaca tggtatctct 16980 caaagagaag tgggtgtaat tccaaaatct aattttggca ggcgctcctg actaaatact 17040 taatctggag atgtcttcaa ggcaggcgga ggttttcagt cctggctgca cattagaagt 17100 cccaggggag ctttaaaaaa ttcccacgtc ctccctgcat cccagactaa ttaatcggga 17160 tctccgaggg tgggaccaca catcagggtt ttgtaaattt ccctgggggt ttggtggggt 17220 tgggggtgga ggcgtctatc ctatggccaa ggttgagaac cactgctttt taaaagactg 17280 tttgcttgtt tttgagatgg ggtctcgctc tgtcacccag gctggagtgc agtggcgcaa 17340 tctcagctca ctgcaacctc tgcctcctgg gctcaagcaa ttctcctgaa aaaggctgtt 17400 ggttattaat gcttccccac agctattcta ttcattgttg catgcttctt acgtgtgcta 17460 ggatgggagc tttaaaggat tacctcattt aatcctcaca accaccttgt gagagaggtg 17520 tcattatccc tgtttggaga gtgagacagg ggcttagcaa gctcagtaac ctgtccaagt 17580 cacacatctg catggggtta gctgctgcta aagctcatgc cgttaatctc catggtacac 17640 ggtgtcctct ccatagcaat cttgcggctg ccttgttaac accaaaaaaa cttgcatcag 17700 ctggtttgac aatttctaga taaagagctc ttttcgggct gctaagaagc ctaatttttc 17760 atttgatttt cttcttgaac tgtgtcacac tcctcattca tttgatatat tcatcaaata 17820 cttattgagc acctgctgtg tgcctggtgt gcagcagtga caccagacat ccaaagtcct 17880 tttcctctta gagcttattc tatctgggag agacagataa taaacacaaa atcagtaagt 17940 cattttatat ggtggtaggt gccttgagga agatgagcca ggttaatggg attaagcctg 18000 gtagggggag ggtgccactt tagctcggaa agggtagcga gacccaaaca atgcaaagga 18060 cccggcccgt ggagatctaa gacaggagga tgccagggac aggaagttgc tggggcaaag 18120 cccctgaggc tggactgagc tcagtgttct aggacgggcg tgggcagtga ggagcagcag 18180 aggaggtgag ctgggagata gcctggggac tctttcttct gcctccttca aaaaataaaa 18240 ctagccaggt gtggtggctc acacctgtaa tcccaacaat ttgggaagct gatgtaggtg 18300 gattgcttga gtccaggagt tcgagaccag cctgggcaac atagtgagac ccctcccccc 18360 atttctacca aaaaatcaaa aaattagctg ggcccggtgg cgtgcgcctg tggtcccagc 18420 tactcaggag gctgaggtgg gagcattgtt tgaacccggg aggtggaggc tgcagtgagg 18480 cgtgattgtg ccactgtact ctagcctggg tgacagagtg agactctgtc tctaaataaa 18540 taagtaaatc tagaacctaa catcttggag tgcagtggca ccaccatggc tcactgcagc 18600 ctcaatctcc tgagctaatc gagcctcccc ttcagcctcc tgagtagctg ggactatagg 18660 cgtgcaccac catacctgaa taatcaaaac ctaacatctt taaagaacat tggcataaga 18720 cttggcaaaa atggcatctt gtccctcatc tcatttagtc caagcgatac aggaaatgct 18780 gccacctcca ttttatagat gaggagtctg acgttcctag aggttcaatg ccctgaaacg 18840 tcaagccttg aggaagttgg agcactggga ttcgaagagc accatccaat acagacccag 18900 aatcaggatg atttgggatt atgcttgtca aggactcagg gcagggctac catacattag 18960 gcacaagaat tttgatagtg ataattactg tgttcattgt cacttcatca tgacagttac 19020 cgtgatgata agaaacctgg cccttcttca cctgacaaag gctttcttcg tttgagccac 19080 tgctcaaacg agactgacca agaataaatc ctcggggctt tggcctttaa aataggaagt 19140 catcataaat gacttgatgt ggtgtgtttc attcttgctt tgcaccagtg gaaaatatac 19200 aggtcaagca tcaaaacatg gcaaatgggg accccaatta ttagagaatc taagttaatt 19260 tttatgtata attaattatt caacaaccct ctcctctcca aaccaataat taatccatct 19320 tttgtatttt aagaccaatt ctgtagtatt ttccatcaat atctatttac tgctagcaga 19380 tatcagctac attctttctc ctttaataga agttccctct ttaggtatta agattcatta 19440 aacaacaata acaaatctac cttgcctccc agggacaatg cacagttctc attcatttgt 19500 tcatttagca gataattttt gaatttccac tgtacagcag ccctgtgctt gtggttggcc 19560 tgttatttga gaagcatcaa ataataatct cattttttgg ctgggtgtga tagctcacgc 19620 ctgtagtccc agcactttgg gaggctgagg cgggtggatc acttgaggat gggcgttgga 19680 gaccagcctg gctaacatgg tgaaacctcg tctctattaa aaatacaaaa attagccagg 19740 tgtggtggca gacacctgta atcccagcta ctcgggaggg tgaggcagga gaatcgcttg 19800 aacctgggag gcagaggttg cagtgagccg agatcgcccc attgcactcc agcctgggca 19860 acaagagcga gactccgtct caaaaaacaa aacaaaacaa gacaaaaaaa aacccaacaa 19920 ataaaataaa taatcccatt tttctccatt tttgagaaag atttctttgg tctgaagtct 19980 ttctctcccc tctccgaggc attacccagt ttaacctttc atgtataata tatatgatag 20040 ttatttaaag tatagcagga caaaatgtat ttgataggag aaaaccttgt ttgctctgtg 20100 ttaagtcctc cagagagcta attagagttt gtgattctaa aaggcaacta tagattcact 20160 tatattagca gttcatgtag attccagtta aggaaatggt ttgtcacttg tgttattgaa 20220 aacacacaca gggcgagcac tgtggcccat gctggtaatc ccagcgtttt gggaggctga 20280 ggtgggcaga tcacggggtc aggagtttga gatcagcctg gccaacatgg tgaaaacccg 20340 tctctactat aaatacaaaa aattagctgg cagtagtggc aggcgcctct aatctcagct 20400 actcgggagg ctgaggtagg agaatcgctt gaacccagga gtcggaggtt gcagtgagtc 20460 gagatcgcac cattgcactc cagcttgggc aacaagggca agactccgtc tcaaaaaaaa 20520 agaaagaaaa cacacacaca aaaaaacttt agtagatctt tcggcatatt attttttaaa 20580 ataaactgat aatggttgat atgattgttc aaagaaataa gagcttttca taaactcagt 20640 ttaaagaaac tttacaggcc gggcgcggtg gctcatgccc gtaatcctag cactttggga 20700 ggccaaggcg ggtggatcac ctgaggtcaa gagttcgaga ccagcctggc caacatggta 20760 aaagcctgtc tctattaaaa aatacaaaaa ttagccaggt gtgttggctg gcgcctgtaa 20820 tctcagcaac tcaggaggct gaagcaggag aatcgctgga acctggtagg cagaggttgc 20880 agtgagacaa aatcgtgcca ttgcactcca gccccagctg acaacagcga gactccatct 20940 caaataaata aataaataaa taaataaata aataaataaa ggagctttac agaaaccttc 21000 tgatgttttt ttcttcttga cgataacatt gccaacactg aatcttacaa agataagaca 21060 agaaagggac cttcagacac cattacatgt aattctggac ttagtggttt aaatccttat 21120 ttttctatga cattaaaaaa atgtatattt taggccaggc acagggctca cacctgtaat 21180 cccagcactt cgggaggccg aggcaggtgg attgcttcag cccaggagtt caagagcagc 21240 ctggggaaca tagtgagacc cctgtcccta cagatttttt ttttttgttt gagatggagt 21300 tttgctcatg ttgcctaggc tggagtgcag tggcacgatc tcggttcact gcaacctctg 21360 cctcctgggt tcaagcaatt ctcctgcctc agcctcccaa gtagctggga ttacaggcat 21420 gtgccaccac acccggctaa ttttgtattt ttggcagaga ctgggtttct ccatgttggt 21480 caggctggtc ttgaactccc aacctcaggt gatctgcctc cctcagcctc ccaaagtact 21540 gggattacag gcgtgagcca ccttgcccag cctacaaaaa gttttaaaaa attaaaaaat 21600 tagttgggca tggaggtgca tgccagctac tcgggaggct gaggcaggag gattgcttga 21660 gcccatgaag tggaggctgc agtgagccat aattgcagca ctgcactcca gcctgggcca 21720 tagagcaaga ccctgtctca aaaatatata tagtatccaa ataaacacaa taattacaga 21780 aaattgaaaa gtgcccataa gcaaaaaaaa aaaaaagaaa aaattaatca cctgcgttct 21840 catcacccag aattaaccat tgttaatatt tttgttatag atccttccaa acttttctcc 21900 atgcttgtga ttgtatttat tatacatgat ttacagggat ataaacgact gtattattag 21960 tcattagaag aactggatta tggccgggca cggtggctca cacctgtaat ctcagtactc 22020 tgggaggctg aagtgagcag atcatgaggt caggaaatcg agaccatcct ggctaacaga 22080 gtgaaacccc gtctctacta aaaatacaaa aaattacctg ggcgtggtgg caggcgcctg 22140 tagtcccagc tactcgggag gctcaggcag gagcagagat acctatctgt tctcaggatt 22200 ttaaggtgtt gcgcggaaat aagaaaaccg tacagtgttt ctcactacaa agcagggtca 22260 ggagatgcaa acaaactgat gtgggggttc caagtgaggt ggaattccag acaggggccg 22320 ggaagacttc gtggaaaggg agaatctgag gtgggttttc taggatgggt aaagttcatt 22380 agaggaagag aagtgcaaca gaggaagttc ggtgagaggt agagggaagg cgttctgatc 22440 atgaaggaaa cactagaaaa ggtatggaga tagaaaaaga taaggcctga ttttttaacc 22500 taccacttaa aaaaaatcct tgaaaagaga tttttaaaac gaatacttgg tgctgacaaa 22560 ggtgaaatga ccgggcgcgg tggctcacac ctgtaatctc agcacattgg gaggctgagg 22620 cgggcagatc acttgagctc aggagtttga gaccagcgtg gccaacatgg caaaactcca 22680 tctctactaa aaatataaaa attagacggg tgtgatggtg ggtgcctgta gtcccaacta 22740 ctcaggaggc tgaggcagga gaattgcttg aacccgagag gcggaggttg ctgtgagctg 22800 agattgtgcc actgcactcc agcctggata gcaggatgag actgtctcaa aaaaagaaag 22860 aaaaggaaag aaaaaaaaat ccgtactgta aactggtaaa ggctttcttt ctggagagca 22920 atttggggca catgcaccag tagccttaga aggctcatgc ttttgaccta attatcctat 22980 tagtggtgag atgattaaag atgtggcccc aatttatgtg aaaggtatgc atcacatctt 23040 cactcataat caggagagtt ggggaaaacc ctagctgtta atagtttatc caaaatccat 23100 atatatatgt gtgtgtgtgt gtgtgtgtgt gtgtgtatgg atttatatat atatataaat 23160 ggatatatat atatatctgg atggatatat aaatatgata tatatatgtg tgtgtgtgta 23220 tatatatatg tgtatatatg tatatatata tgatggaata ctatttagcc ataaaaagga 23280 atgaattaat ggcattcgca gtaacctgga tggacttgga gaccattatt attttatttt 23340 atttatttat ttttgagacg gagtctcgct ctgtcaccca ggctggagtg cagtggctcg 23400 agctcagctc actgcaagct ccacctcccg agttgacgcc attctcctgc ctcagcctcc 23460 tgagtagctg ggactgcagg cgcccgccat cacgcccaga taactttttg tatttttagt 23520 agagactggg tttcaccgtg ttagccggga tggtctccat ctgctgacct catgatccac 23580 ccgcctcggc ctcccaaagt gctgggatta caggcgtgag ccaccgcgcc cagcgagact 23640 gttattctaa gtgaagtaac tcaggaatgg aaaaccaaac atcgtatgtt ctcactcata 23700 agtgggagtt atgctatgag gacgcaaagg cataagaatg atacgataga ctttggggac 23760 tcagggaaaa ggtgggaagg gggtgaagga taaaagatac aaattgggtg cagtgtatac 23820 tgctcgggtg atgggtgcac caaaatctca taaatcacca ctaatgaact tactcatgta 23880 accaaatacc acctgttcct caataaacca tggaaattaa aaaagaaaaa agaaaaagta 23940 ccctggaaaa aaaatttctc cctggccagt cacggtggct catacctgta atcccagcaa 24000 ttcgagaggc tgaggcagga ggatcacttg agcccagtag ttcaaaacca gccagtgcaa 24060 catagtggga ccctgtctca aataaaatct aaaaattagc caggtgtgtt ggtgcatgtc 24120 tgtggtccca gctactcagg aggctgaggt gagagtattg cttgagccta ggaggttaag 24180 gcggcagtga gccgtgattg tgccactgcc atccaacctg ggcaacaaag caagaccctg 24240 tctcaaaaaa aaagaaaaaa aaaacctctc tattcgcctt ttaagaatac ctgggcttct 24300 ctgtgtacac ttaagcttca ttggagtctt tagacttttt ttttgctgta tctgtccagt 24360 taccaagtcc cagcttctac tccatgctcc ccatgctctc ttcctatttt attttccatg 24420 actgcctcgg tataacttgt gctcaaccaa actggactac tcaattccct gcattttctt 24480 ttttaaagtt taatcaaaaa aaaaaagaaa actggctggg cacagtgggc ttctgcccac 24540 aatctcggtg ctttgggaaa ctgaggcagg aggattgctt aaggccaaga gttcaagacc 24600 agcctgggta acatagcaag acctccatct ccacaaaaaa atttaaaaat tgactgagtg 24660 tgatggtgtg cacctagtcc cagctgcttg ggaggctgag gcaggagaat tgcttgagcc 24720 caggagttcc aggttatgat gagctatgac tgtgccaccg cactccagcc agggtaacag 24780 agtgggactg tctcaaaaaa caaaacaaaa tccctaatat aatctcagtg tgccttttaa 24840 gtatgccata tatatatata tatatatata tatatatata tatatatatc acattttctt 24900 tatccactca ttgattttca tgtagttcta atcgtagaat tcatacattc tttctatctt 24960 ccatctttca cataacatca caaacatttt ctaggttgcc atattgtctt catagttact 25020 taaataatat tccatcaagt agcacaatca tttatttcac tagtcctcta actgtagaca 25080 ttttggttgt ttttgaaact taataatgta aataacaccg tgataacaat gtttatgtaa 25140 attcatattt tggattatct ccttagggtg gattcccaga agtcacatta gtaggtcaaa 25200 gagtatgagc ctattttcaa ggctcttgtt ttattacctt ttaatttcca cttgcctcaa 25260 tattgctggt ttgctccctt atgatcacca gagttactcc gtcggtccaa attctttacc 25320 ttccgaaact gggaaggcca tgactcaatg ttatatatat agtaaaggct actataacct 25380 tccccagaat tttccaagcc agtggtctct aaagtgacct ttggctgtta aaatctgaat 25440 tcagagggtt catgagactc agtgttgttg tagaatttaa gctccttaat ttgccacgtt 25500 gtttagacac cacttaatac tttattgcaa atgacttgtc aacgcctctc acctacaaac 25560 ttcatcctcc tacaaatata cctcctgcta atcaaatgag gctacagttg agtctttaag 25620 tttcagtaga aagatggccc ttcctctggg gtaggcgcat gctcttcatg ctgaagctca 25680 gctgaaaagc ctcctgctga gttttctgcc tctttccctc ccactgcaca caccccaggg 25740 tgttggcgcc acttcaaagg gagcctgtgg atgaagaaaa cacaggtaaa ggcagagggc 25800 tcataagggg gccataaatt taaaaagtta agattcctgg cactatcaac tctcacttgt 25860 tttcaaatat gcatatggag tggatattcc agttttcatg tctgtgttgt tgtttttaaa 25920 aaaagacctt tcaaagaact gtgcattttt tacaggctga caggctgtgt ttggtgttaa 25980 actgtcaggg ctgactggtc acttggaaag ggcaagggct gaggtgcatg caagtgtcgg 26040 ctggttactc acagacacag cagccccttt taccccggag agagttctgt ttgctggagc 26100 ccttattctg gccagcagtg tcacaaatgc acactgtaag acatagacag tcttggaaag 26160 aaagggaaac tggctttaaa aattcttact ccttctagca aagcaattca tctttggcta 26220 taaagaataa cacagccagg tgcggtggct catgcttgta atcccagcac tttgggaggt 26280 caaggtgggc agatcacttg agtctaggag ttcaagacca gcctgggaaa catggtgaaa 26340 ccccacctct accaaaaaaa aaaaaaagaa agaaagaaaa gattagccag gtttggtggt 26400 acgtgcctgt agtcccaggt actcgggaag ctgaggtggg aggatcgctt gagcctggag 26460 ggcggaggtt gcagtgagcc gagatcatgc cactgcactc cagcctgggc aacagagtga 26520 caccctgtat caaaaaaaaa aaaaaaaaag aacagtaaca cattattaga aatgagcatt 26580 ctgaggccag gcacggtggc tcatgcctat aatcgcagca ctttgggagg ccgaggcggg 26640 tggatcacaa ggtcaggaga tcgagaccat cctggctaac acggtgaaac cccgtcttta 26700 ctaaaaacac aaaaaattag ccgggtgcag tggcgggtgc ctatagtccc agctactcag 26760 gaggctgagg caggagaatg gcgtgaaccc ctgggaggcg gagcttgcag tgagccgaga 26820 tagtgccact gcactccagc ctgggcgaaa gagcgagact ccatctcaaa aaaaaaaaaa 26880 aaaagaaaga aacgagcatt ctgaaatagt cttccatatg atgcttttga caattcagca 26940 ggaaaataaa ggatgtaaga aatgaatgca tatgttaggc ctcttgttga cctgtggact 27000 aaattgtttc tccctgcaga gatcagcaag gacaactcct gcaaagaaaa ctgtacttgt 27060 tcctcctgct tgctccgggc ccccaccata agtgacttgc tcaatgatca ggacttacta 27120 gacgtgatca ggataaagct ggatccgtgt cacccaacgg tgaaaaactg gaggaatttt 27180 gcaagcaaat gggggatgtc ctatgacgaa ttgtgcttcc tggagcagag gccacagagc 27240 cccaccttgg agttcttgct ccggaacagt cagaggacgg tgggccagct gatggagctc 27300 tgcaggctct accacagggc cgacgtggag aaggttctgc gcaggtgggt ggacgaggag 27360 tggcccaagc gggagcgtgg agacccctcc aggcacttct agagctcttc ttcttccttc 27420 attggcctct ccggatgttg aaacaaccac aggtcaagaa ggaatgtgaa tctgttgttt 27480 tataagagtt taggacaagg acgtggaaca gtggacactg gttttcccca aagctggcag 27540 ttttgtggag gggtagcttg tttcggtggt ggatctctgt ttatttttgc acatctgtta 27600 taatttaata ttcaaatctg gaattaagaa aacatatttt ctagtatcct ctaagggcca 27660 aagtcctaca atcggaatgg attcatgcca cgttgaagat aaaattatcc tctctctgaa 27720 atacggtaaa gatttaaata ggtcctgaga ctgttgatag ccccagacat acccacagca 27780 ttatatgtaa catctctcct gatcagtgcc attcccacgg tttcaaagaa aacagctaca 27840 aggaatgctt acctgagtgt ctgcagcacc ctccacttct ctcctaggca atgagaccca 27900 gtggctagaa attcaccatg tctattctca agatccatgc cagggagctc tttgactctc 27960 gtgggaatcc cactgttgag gttgatctct tcacctcaga aggtctcttc agagctgctg 28020 tgcccagtgg tgcttcaact ggtatctatg aggtcctaga gctccaggac aatgataaga 28080 ctcgctatat ggggaagggt gtctcaaagc ctgttgagcc catcaataaa actattgcac 28140 ctgtcctggt tagcaagaaa ctgaacgtca cagaacaaga gaagattgac aaacttatga 28200 tagagatgga tggaacagaa aataaatcta aatttggtgc aaatgccatt ctgggagtgt 28260 ccctcgctgc ctgcaaagct agtgctgttg agaagggggt tcccctgtac caccacatcg 28320 ccgacttgtc tggcaactcc aaagtcatct tgccagtccc ggtgttcaat gtcatcaatg 28380 gcagttctca tgctgtcacc aagctggcca tgcaggagtt catggtcctc ccagtcggtg 28440 cagcaaactt cagggaagcc atgcccattg gagcggaggt ttaccacagc ctgaagaatg 28500 tcatcaagga gaaatatggg aaagatgcca ccggtgtggg ggatggaggc gcgtttgctc 28560 ccaacatcct ggagaataaa gaaggcctgg agctgctgaa gactgcgatt gggaaagctg 28620 gctacactga taaggtgatc gtcagcatgg acgtagaggc ctccgagttc ttcaggtctg 28680 gaaagtatga cctggaattc aagtttctcg acgaccccac caggtacatc tcacctgact 28740 gtctggctga cctgtacaag tccttcatca aaaactaccc agtggtgtct actgaagatc 28800 cctttgacca ggatgactgg ggagcttggc agaagttcac ggccagtgca ggaatccagg 28860 tagtggagga tgatctcaga gtgaccaacc caaagaggac agcctcggcc gtgaatgaga 28920 agaagtgcaa ctgcctcctg ctcaaagtga accagattcg ctctgtgact gagtcccttc 28980 aggcgtgcaa gctggcccag gccaatggtt ggtgtgtcat ggtgcctcat cattctgggg 29040 agactgaaaa taccttcatc actgacctgg tggtggggct gtgacctggg cagctcaaga 29100 ctggtgcccc ttgctgatct gagcgcttgg ccaagtacaa ccagctcctc agaattgaag 29160 aggagctggg cagcaaggct aagtttgccg gcaggaactt cagnaacccc ccagccaagt 29220 aagctgtggg caggcaagcc cttcagtcac ctggtggcta attagacccc tccccttgtg 29280 tcaactccgg cagctcaaga cccccgagca acatttgtag gggccgctgc tagttagcta 29340 cccttgccca ccgccgtgga gttcgcacct cttccttaga acttctacag aagcaggttg 29400 cagtgagccg agattgcgcc actgcacacc agtttggaga cagagtgaga gtccgtccca 29460 gaaaaaaaaa aaaaaaaaaa gaacttntac agaagccaag ctccctggag ccctgttggc 29520 agctctagcc ttgcagtcat gtaattggcc caaatcaccg gagccacgtg accctccagt 29580 gtcatctccg gggtggccac aggcaagatc cccagtgatt ttgtgctcaa aataaaaagc 29640 ctcattgacc catgagaaaa aagaaaacag caatgagaag tgaccctgtc ttgttggttt 29700 attacttttt ttgttataaa gtactttggt gaattaacag gatgctagta ttacatggtg 29760 atactcttca gaacacctgc cccatctttt ttatgcaagt atgtttacaa tcagtggact 29820 atcagtaatg tcatttgctc aaatattttt taaagaccta cagaaactga tggttattgg 29880 gaaaacagtc aggaagtagt gaggtaatca aggccatggg aatagtgttt gacaaagaga 29940 gtactccaaa tcccttttgg ttacccagga ctttaaaaaa gagagtactc catcacacct 30000 gtaatcccag cactttggga ggccgaggcg ggtggatcac gaggtcagga gatcgagacc 30060 atcatagcta acatggtgaa accccgtctc tactaaaaat acaaaacatt agccgggtgt 30120 ggtggcgggc gcctgtagtc ccatctactc aggaggctga ggcaggagga tggcttgaac 30180 ccaggaggcg gacttgcagt gagccgagat agcaccactg cactccagcc tgggcgacag 30240 agcaagactg tgtctcaaaa aaaaaaaaaa aagagtgctc caaatctcct ttggttaccc 30300 gggactttaa aaaatttaat gtgatagtta ggccgggtgt ggttctcacg cctgtaatcc 30360 tagcactttt ggaagctgag gcgggtggat catttgaggt caggagttgg agaccagcct 30420 ggccaacatg gcgaaacccc gtctctacta aaaatacaaa aattagccag gcgtggtggt 30480 gggcgcctgt aatcccagct cctcgggaaa ttgaggcact agaattgctt gaacccagga 30540 ggtggaggtt gcagtgagcc gagattgcgc cactgcactc cagcctaggc aacagagcga 30600 ggttccatct caaaaaaaaa aattgtaata ataataataa caatgtaata tttacttttt 30660 catcctttat ataaggctga gtgcttcacc cctgagatga agctcagtta agaaataaat 30720 gaaaatcccg taacctattg gtgaaaggta accaccccca gctcctacta gcccaactta 30780 aaacaggacc ccatcacact acacagcagt ttagccaaga aaagggggtc tttatgtgga 30840 cactgggagg gaagggattc cttcaaatcc aaactttaaa ggattttaaa caaatgaaac 30900 atttggttca aagaatagct gatgttttta tttgatgatt ttggagaaag gaaagtgtgg 30960 ggcataatgg ggtttgttat tggaaagatc agattttcta ggtaatttgg gtggagaaag 31020 acaaaaggca aagctttgac tgacaattcc atgaaagtgc tatttggttt tggttatggg 31080 cttagaaaat taagacactt agttcaattt ggaaggattc tgtataagtc cctgattaaa 31140 ataagcaaaa atgatgaata acactgattc agtgcaaccg aaagattagg attaactcaa 31200 aagaaagtta ttttctaaac caccgtgatt ttttccactg acaattacag cggttttcat 31260 taggttgctg acacatgaag tcagcctcac catcagttgc aaactctaaa ctagcaaaat 31320 ctattacaga gacatactta tcacttctga tttagtgcta atctcaccca gctcatcttc 31380 tcttgtcaga tttatgagat aaatgtcaga tttatcacca gatatattga aagtaacagc 31440 cagtaataaa atgtgagatt ttaaaaaata gattctttgg caaattggtg ttcagtgagg 31500 caattattaa acatttttgt cagccaggtt ccaggcactg tacagaagct gttaggagtt 31560 ctcaccatct acgaatttga tttgatgtat tgtattctca ttaagctatg tgtgacacat 31620 tgtcatttat tagcccagaa tttaaaaagc tgtggttgtt tagtgttggt ggtagcagac 31680 cccagcagtc tgatggtctg cactccttcc atcctgccac cccctgggga tgcaaagact 31740 ggatctcagg gtgacaatct tcttgcgcac gactgcctgg ccaagtgcct ccagaaagcc 31800 ccttccttcc cccatttcca cccaggccca cttgtcacct cagcctaaca ccagcctgca 31860 cagtctacgg ccaccatcca ggcagtggga gagggaaagg ggaggagggt ggaagggaaa 31920 acccctttct atacctctcc tcagcctgct ctttcctcct cccacctctg agcctccgcc 31980 tcccccagac agagacagaa aagatggaag aacaggtggg acctccaccc ccaccccaag 32040 ccttcatccc ggtggagggg gatgggaaga tttctctcat ttcaagagac tcctccacct 32100 cagactgaca aaaggcagag gcctggcaag aagaaagggc accctgggga agaagggcat 32160 tgaaatagca cctgccgggc cgggcacggt ggctcacgcc tgtaatccca acactttggg 32220 aggccgaggc gcgtggatca cggggtcagg agttcaagac cagcctggcc aagatggtga 32280 aaccccgtct ctactaaaaa tacaaaaagt agccaggcgt ggtagcgggt gcctgtagtc 32340 ccagctactc cggaggctga gacagggaac tgcttgaact gggaaggtgg aggttgcagt 32400 gagccaagat cgtgccatgc actccagcct gggcgacaga gtgagactcc atctcaaaaa 32460 accaaaacag aaatagcacc tgcccccacc ccctgcccgc cctccttccc gcccccgtcc 32520 tttcctagac ttcactcaag tcctctgctc agaggaagcc ctgctctact gaaagccaca 32580 aggccattct cggtggcctg ggacagcagc ccaagacgtg ggcttctaac tgcctccgaa 32640 ggggccacag cagcaaacat aaataaaaat agtaaaatgt tcttaaatta taaatttaaa 32700 atttggaaaa tttagtgagc acagcttcta gggggcatgt ttccaaaatt ccaaccacaa 32760 aagtgcagtc tcaaaactga ctgtaaaccg aacataccat ctcatctcag acacagctat 32820 tgttcacgag tgtcagtgga actttcctcc cttgagatgg accaaaaacg tcaagcaaga 32880 tgacatttgc tgatttgcag gcttcaggca gataagatac gggcagagtt gagtgtgcgc 32940 ctttaccctt aaattcagga atagcaggaa cagcaggaaa aacgtaggac cacagcgtac 33000 gtcccacttg tctttcattt tgatatcatt atttccagag tcctgattgc tagtcatgtc 33060 taacactgga tttattatca tctcattgct agcatggcta ggaaagcttt gaacatcctt 33120 atcattctat tttaattcct attataattg catggggaag ttccagggtg gaaaaatttc 33180 cttttctttc tttttttttt tttaatagga gagttggctg ggcacggtgg ctcacgcctg 33240 tgatcccaac actttgggag gccaaggcgg gtggatcacc tgaggtcagg agttcgagac 33300 caacctggcc aacatagtga aaacctatct ctactaaaaa tacaaagtta gctgggtatg 33360 gtggtgcaca cctatagtcc cagctactgg ggaggctgag acaggagaat cacttgaata 33420 cgggaggcag cagtgagcca agatcatgcc actgcactcc aacctgggag agacagagtg 33480 ccatctcaaa aaaaacaaac aaaagagttg atataaattt gctgttataa tttgactgta 33540 ctgtttcttg cacatgttga catctgtaat gactggagtt tatgaaaatt tttgatgagt 33600 aggagcatac cattaacaga gagaaattta atcaaaagat ttttaaagtt ccttcagagt 33660 ccagactttg actaagtgta gtatgattta tatctatgtt gcatacaaaa atatcaaaca 33720 gtaattccca actgaaatac aagtatcaat caattgtgta acaatgcaaa atcatttaat 33780 ttaaagttaa tttatagcaa atgagtactg taatagcata agcatgccga tactttacaa 33840 aggagagagt ggaaaggtag gatattataa ctaattgatc aaatcattgt taaaatttaa 33900 gtttattaat acttttactt ctgtccgtag ggatccatgt taaattgggt atattataaa 33960 cttaactgct aatgatgagg tccttttgct attagaaatc tattttttat ttttctttat 34020 tattttttga ggcagggtct tgctctgttg cccaggctgg agtgcagtgg tgaaattata 34080 gctcactgca gcctcaacct cctgggctca aggaatcctc ctgcctcagc ctcccaagta 34140 atggaaactg cagtcgtata caagcacacc cagcaatttt tttttttttt tttggtaaga 34200 tggggtttag ctatgctgtc caagctggtc tcaaactcct ggcctcaagt gatcctccca 34260 ccttancctc caaagtgctg ggattacagg cgggagccac cattcccagc ctagaatgaa 34320 atatctttag ctaaattaca gggctggatg tggtggctca tgcctgtaat cccagcactt 34380 tgggagactg agggcgggag ggtcacttga gatcaggagt ttgagaccac cctgggcaac 34440 acagtgagaa ttctgtctct attttaaaaa gagaaaaatc tagggtatat tctcttaaac 34500 aaaactttca tctataatgg tagttgatga ggtcctatgt aatatgcatt tccttggttg 34560 caatagcaaa ttactacaca cacagaaagg aaagccacac tccccgacac atctacacac 34620 aggaggactc acacaggagg gagactcaaa gaaggcacgt gacttttaca ttgttagggc 34680 ttacatggtc ctgggatttc ccaccagtac tcaaaagatc aattgtatga acaagtcacc 34740 tatttttacg gcactaaata attattattc aacaacatgg aaaatatgtg gtagcagacc 34800 tggattttcc ttaagagtta tttttatgtg gtactgcccc ctgctggaat ataacatcta 34860 tacacatcct ttctggctgg gctgacatcc taaaaccagc ccaggaccag ccttttatta 34920 atattaattc ttggccaggc gcggtggctc gcctgtaatc ccagtacttt gggagtccag 34980 ggcgggcgga tcacgaggtc aggagttcaa gaccagcctg gccaacatgg tgaaactccg 35040 gctctactaa aaatacaaaa cttagctggg catagtggca cattcctgta atcccagtta 35100 ctcgagaggc tgaggcagga gaattgcctg aaccgggacc cgggaggtgg aggttgcggt 35160 gagccgaaat cgtgccactg cactccagcc tgggctacag agcgagactc cgtctcaaaa 35220 ataaataaat aaaaattaaa attaaaaaat aattcttggt tgtatgctaa aagccttgca 35280 agtagcccca ctggaagata ggaagagtgg ggctgtttta caaatgagca catataagca 35340 gaacgaggcc gccataattg aaatgaaggt ccccgtcccg tggatgtgtt catcgctact 35400 tcaccctgtc attcggatcc aatgtgtgac cagccagctc caataacagt tccatactct 35460 gggaattatt tttaacactc ggcaggatgc tttcttcctg tagttttagg cttagccctt 35520 tgtgcacttt tggtctcttt ccctttcaat ttagcatcca aggaagcggc tgtgaccaaa 35580 ggtagctgtc atgttaaagg acaaagttca tagttacagc aaatattgac ccagagcact 35640 atccttgccc cttcctctat aatgtgcaat gcaaaaatat gttcttttaa gtacaatatt 35700 aataagtaag gtctaggaga ttttcttccc ccttcctttc tcttttagat gagtaaatgt 35760 tttatctagt tttgaggaga ctatccttct tatcacatct ctttccactt ctgctctcct 35820 tgttttataa ttttcctctc ctttgggtcc gtgtcattat ttcgtgtcgc ttgttttcga 35880 gccatgcact catttatcaa atcagatttc ctccgtatgc cgacggcctt cctctccctg 35940 ccacgggctt cctttttccc tgactatgca gaagcaattt gttcgcttgt gtttcttttt 36000 ttttttgaga cagagtctcg ctctgtcacc caggctggag tgcagtggcg acatctcggc 36060 tcactgcaac ctccgcctgt caggttcaag caattctcat gcgtcagcct ccagagtagc 36120 tgggattaca ggtgtttgcc accaaggctg gctaattttt agtagagacg gggtttcacc 36180 atgttggcca ggcttgtctc gaactcccaa catcagttga tccacccatc tcggccttcc 36240 aaaatgctgg gattataggc atgaaccacc gcatctggcc ttgtctttca tccttaatga 36300 cactttagtc ctaataatgc taaaatcatt ttctactctt tgaattgaaa cacagcttat 36360 ctacatgagc ccaaggcagt agcaacattc acctccattt cttctctgat ctctaccttc 36420 tgaaccctgt ggacttggtt gtaaatggat gagggcaagt cttgcttcct tcccctgtgt 36480 ttacagagga tcgtggctga gatgctgggc cacactctgg gcctgctggc acccctgggc 36540 cggtggctgc tgcccctcag ggtgctcacc acctagacca gaagaaccaa ggtgagggag 36600 agcctgtttt ctttcttcct gtggctgcgg gggctgtgag gcatgggtct agtggctgtg 36660 tttagctggg gatgcctcct agaaatcagc tccaccgttg aagagatcaa agcaatgcac 36720 agtgccactt gaaatgaaac gattgagctt atcagcgctt ttgcaaatgt acaagagggt 36780 agctcccccg gacatcctga actgagccat gctcttctat tttgtgtaac agcccagtga 36840 cccctgaatc ttcccctgag gcaggtcccc gaagcttcat ggaggatgtt cctcagctga 36900 ccaaggtgag gctcttgagc tcctaaatct ttgtgatact gtttatacat ctttgtgctg 36960 tactttttaa gctgacttcg tgttatcacc tgtatgattt tatgttttgc ttctaaataa 37020 gtacagatta ttttaaactc taataatggg tgctacaaaa ttaaagatta tgtcaatcac 37080 tgtctctgat gagttatttt atgtagattt caacacaatc attgattcat gtgtactctt 37140 ggtcagtcat cagtcatctg agtacctagt gggtttccaa aatgggtcct ggatgctggg 37200 gatgcaaaga taagcaacac atttctatcc tcaacagcct gtagatgagg gagaatcact 37260 gcggacaatc agggaagtta ccggagagag cagtgcacat gtggtctaga aactggtgga 37320 acaaagttga gaatcactga actaggagga aagacaggtc actgacaatc caaggcacag 37380 tgactcacac tctaatctca gcactttggg aggccaaggc aggaagatcc actgagctca 37440 ggagataaag accagcctgg ctgacctatg gagacccttc tctaccaaaa aaaaaaaaaa 37500 aaaaaaaaac attagcctgg catggtgggg tgtgcctgtg gtaccagcta ttcaggaggc 37560 tgaagtggga ggatcgcttg agcccgggag gtcaagactg cagtgaatca tgatcacacc 37620 attgcactcc agcctaggga agagagcaag aaagaccctg tctcaaaaac agaaaaaaat 37680 ccagtaaaat gtttcagatg ttgttaaagg tgatttcact gttacttttc acctctcctc 37740 attttacatc tctgacctat gcttgtcctc tgacttgcca gacattccta gctatggact 37800 tgatgtctcg acatggaggc tcacaggcac cccaaactca gcctgcccta agctgaaccc 37860 atgatctttc cttccaaact tgtttctcac cagagttccc atcttatcat ccacctagtt 37920 gttcaagtca tccttaagac ctccctctcc ttcactgtct attctacctc cctaatatct 37980 cttaaatcct tccctcctct cccacctcac agccaccatc ctaacctaag cagccaccct 38040 ttctcaccct ataatgacct cctggctgtt ctctatagag ttggtgaatc ctttcgtctt 38100 cagcctgaac cccctttcga gggattctta tatatataca tagatataca caaatatata 38160 tgtacatatg tacatatgtg tgtatatatg tacatatgtg tatggatata catatgtaca 38220 tatgtggatg tacatatgta catgtgtatg ggtgtacata tgtacatgtg tatgggtgta 38280 catatgtaca tatgtgtatg ggtgtacata tgtacatatg tatatgggtg tacatatgta 38340 catgtgtgta tgggtgtaca tatgtacatg tgtgtatggg tgtacatatg tacatgtgtg 38400 tatatatgta catgtgtgta tgtacgcatg tacatatgtg catgtatgtg catgtgtatg 38460 tgtgtgtatg tacacgtgtg catatgtgtg tatatgtgta cacgtgtacg tgtgtatata 38520 tatatatata tatactggct ggagtgcagt gggaaagttt tggctcacca caaactccac 38580 ctcccaggtt caagtgattc tcctgcctca gtctcctgag tagctgggat tacaggcgtg 38640 caccaccatg cccagctaat ttttgtattt ttagtagaga cggggtttca ccatgttggc 38700 caggctggtc ttgaactcct gacctcaggt gatccaccca cctcggcctc ccaaagtgct 38760 gggattacag gcgtgagcca ccgtgcctgg ccggattcct atcttgaaga cgaagcccca 38820 gaccatcgac acggcctcaa ggccctgcat gacgcctcct gccccaacac ctcgtgtcat 38880 cttgctctcc tctcccgcag ctcctgaggc tttagccacc ctggaattcc aagtccccat 38940 ggtcattttt ttttcctgct caagatatca ccatgtgctg tcccctctgc ccttgtctac 39000 acccacgtgt ccttctcccg ccccggccac actcatgggg cacactgtcc ttccctggct 39060 aatcctccca cactcgatac cactttctct gggatattgc acccgatcct cagccgcagt 39120 tgtcttccta tgacccactc ccacactctc gccacaatgg taattgtttg attcctactt 39180 gttgtccctg tgagactgca aaccccagag gacaggggcc ctgggttctc cttcgcctct 39240 ggatcatcag cactaactga atacctggcc tagaagagat gctaacgatg ctgaatgaat 39300 aaataagtgg aaagactctc agtaaagcaa aacctttctt taccatttta tggccgtcaa 39360 ggaggaaaac acattatcag tggaaaacgc aaaatgaggg gatttgctta gcaaacgatg 39420 aattcctctg gcaccctggc agccttggtt tcttttgatg aggtccaccc ccttccatcc 39480 atcttctggg cttaagagat caaagcaaaa catgctgtgg aattcgatac tggtgcaggt 39540 tgcacaacat tgtgactgaa ctaaaagcca ctggatggta caactgaaaa tggtgaattg 39600 cgtgttgcat gaattataac ctaactgggg aaaaaaaggc ttaaaaagag acaaagcttc 39660 ccccacaatg gaaaggaagg tataatagaa acagcagctt tcaaaccttg gcaggataat 39720 gaaaccccgt ttctattttt aaaaattagc tgggtacagt ggcacgtgcc tgtagaccca 39780 gctactcggg aggctgaggc tggaagatcg cttgagccca ggagttcaag gctgtggtaa 39840 actataatca cactactgca ctccagcctg ggtgacagag aaagaccctg tctcaaaaaa 39900 ggaaggaaag aaggaaggaa ggaaggaagg agggagggag ggagggaagg agggaaggaa 39960 ggaaggaagg aaggaaatag cagctctgag cttagaaata ggagtctatt tctaagtggg 40020 agatggggag aaggagggaa ctggggaggt gaggaagaag caggtattgt caccagtgag 40080 gactgtgctg ttgtgagccc agctaggcaa ctggcaattc cattctgtta gtgacagcta 40140 caataaccca aagccctctg gagccctgct ttcctctgct ctcttcgtgg cttgactagg 40200 agctgaagat cctgtccctc ttagagcatt ggggcggccc accccacctc caccctcctc 40260 cacctgctgc ctcgaggccc ctcccactcc cggggtagac aaaacagttt agaggctgaa 40320 gtcaccgggg ctgtaactgt tggatttgca catgtcatag aaaatcatca tatgttttgt 40380 gtggactcca tgcataacaa caagagaacc aaccagaccc catagacaga agggagtgtg 40440 aattggagac aaaatttaaa ttatgagttg ccttctattc agatttctcc catttttaac 40500 aaaaaggagc ccaaattcct aaatgttatg gttgtttgca gcaacttatc atctttctcc 40560 tttccttcat agccaaggtt tttgaaagag ctatctgagg ccgggaatgg tgactcacgc 40620 ctgtaatcct agcacagagg ctgaggtgag tggatcacct gaggtcagga gttcaagacc 40680 accctggcca acatggcgaa atcccgtctc tactaaaaat acaaaaatta gccgggcatg 40740 gtggcgtgtg cctgtaatcc cagctactca ggaggctgag gcaggagaat cacttgaacc 40800 caggaggtgg aggttgcagt gagccaagat ctcaccactg cactccagcc tgggcaacag 40860 agtgagactc catctaaaac aaaaaaaaaa gtagctgtct gttctttctt ctcgaactct 40920 ttttcccgct ggagtctgtg acctgctgcc gtctgcctca agtgagaggg actagcagat 40980 ctggtgaatt accttctaat gcccgtaccc tgcccatacc agcttcaatc tgtatgtaga 41040 agcttagctt gctccatgca tggcctccag catccactgg tcacaaaata acacaaaata 41100 gcatgagaga gaatggtcgc atggagcgga ggagctgctg agactgaacc caagccaggg 41160 ctactgctgg gtggaactgg acatgcccag cccatgggaa agtcttccca cagaagtcat 41220 atttgcaggg gtctcccagg agacagcaca ttctgagcaa aggagtgagg cagagataac 41280 tattcaggaa ccaagagact cgctggaaag aagcagagat tttcagccca gcgtagtgga 41340 tgtttcttga atcttcccct gtggatgccc caaaccttga gatccttcca acaaatagca 41400 cactactaac aaactgtgac tcaaagagag ggaaacatgg tcccctgctc tgtcacaaat 41460 cactgtgaag ctttggcacc ctgactgctc aggtggccac caacacagaa ggaccacgaa 41520 tggctgagtc aggaagtcac agccgtgtgg ctggaagagg ctctgccttg ctctgggaga 41580 aatgcctatc cccaaggaag ccttagtatc catgggagag aaacactgta gcaatggccc 41640 ccaggactct cgggaagcca cttctggtgg gaggggactc aaagggtgct gggggacctg 41700 tgtctgcatc tggaagtgag gagccaggaa aattttcttt cagtttcttt cttttttctt 41760 ttcttttttt tttttttttt ttgagaaagg gccttgccct gtcgctcagg ctgaaacata 41820 gtggtgcgat ctcggctcac tgcaacctcc acctcccagg ttcgagtgat tctcctgcct 41880 cagcctcccg agtagctggg actacaggca tgcaccccca cccacgccca gctaattttt 41940 gtatttttgg tagagatgtg gtttcgccat gttggccagg ctggtctcga actcctggcc 42000 tcaagtgatc ctcccgatgt gctgggatta caggtgtgag ccaccacgcc cggcctcttt 42060 ctgcttcatt taacattaat ggtcatccca cagcatggtg ctgtgcacct gtagtcccag 42120 ctactcaggt ggctgaggtg ggagaatcac ttgcgttcca gctgtagtga gccttgattg 42180 tgtctgtgaa taaatgccac ttctctccag cttgagcaac atagggagac tgtctcttaa 42240 aaaacaaaac aaaacaggct gggctcggtg gcccacgcct acaatcccag cactttggga 42300 ggccaaggca agaggattgc ttgagcccag gaggtcaaga gcagcctggg caaaataggg 42360 agaccccatc tctacaaaaa gataaaaaat aaaaaaatta actgggcatg gtgatacacc 42420 tgtagtccca gctactctgg aggctgagat aggagtattg cttgagcctg ggaggtcgag 42480 gctgcagcga gccatgatca tgccactaca ctccagtcca ggcagcagag tgagatcccg 42540 cctcaaaaaa ataaaacaaa acaaaactca tctctccctt ggctcctgag actacaatcc 42600 ctcacggttc ttttctactt ctctgttttt ctcttcttgt ctcccttttt ttctggtctc 42660 tctgtcaccc aggctggagt gcagtggtgt gatcatagct cactgcaacc ttgacctcct 42720 gggttcaaga gatcctccca cctcagcctc tcgagtagct aggactacag gctcacacca 42780 ccatgcctag ctaatatttg tagattttgt agagatgggg tcttgctatg ctgtccaggc 42840 tggtctcaag ctcctggcct caagtgatcc acccacctca gccacccaaa gttctgggat 42900 tacaggggtg agccaccgcg cccagccgat aattgttgaa aaatcatttt cagttaaggt 42960 atccagtcaa ggtcagaaaa tgagaaaatg ttaaaaaaaa aaaagctata agtaaaacag 43020 attcagtcgg gacatgatgg ttcacgcctg caatcccagc actttgggag gttgaggtag 43080 gataatcact tgagcccagg agttcgagac cagcctgggc aacatagcga gaccttatcc 43140 atacaaaaaa atttaaaaaa tacccaggca tggtggcata ctcctgcatt ccctgctaat 43200 tggatgggtg aaggggagga tcccttgaaa taggagtaga ggtgcaggaa atatgattgt 43260 gccgtgtaat ccagcctggt tgacagagca agatgttccc cacccccctg aaaaaaaaaa 43320 aaaaacctaa atccaaattt taaaagtttc cttgactctt caacttgctc accctccacc 43380 aaataaaata actacgaagg aggcttattt tttactattt ccagggatac gatatatgtt 43440 tgtcctgaaa atatacatca tggctttact caagccacag tgatgaggcc tcattgtcac 43500 tgtagcctaa ttacgatttt ataactccat ttaaaattca atttaaacac agtttaaaaa 43560 ttcagtccaa gtcaaacatg ctctcagtag ctagaagcaa aactctgttc aggtccttga 43620 tggatctatt tgtactttct ttcatgaaaa cagaaagtcc ttttttacac accatgcaac 43680 aggaaaattc ataacggaca ttgttttacc tgttcttggc aaagacaagt gagctcttaa 43740 caagcaaggt aactatggag atgatgtttt gctccaagtt aacacttaca tatttaatta 43800 gaaagatttc aaaggtgggc agattcactg gaaagtttcc aaaagcttca cttgttcaac 43860 aaataatgtt agagagggag caccgtgccc tcgggcccct aggaattagt tccacatggt 43920 ccggtcctct gtccagtgtg cccagcatcc acttgggaga acagcatggc cttctgtcca 43980 gggcagccca cgccagcact gcctgccctt tcaggcccat ggctcccatt aagtgccatt 44040 tcgagcatac ttagccaagt ttccctacca tggccaacaa agaggttgtt caaaaatgct 44100 tgtcaggtcg ggcatggtgg ctcacgcctg tagtcccggc actttgggag gctgaggcgg 44160 gtggatcacc tgaggtcagg aattcaagac cagcctggcc gacatggtga aaccccgtct 44220 ccacaaaaat acaaacatta gttgggcatg atggcgggtg cctgtaatcc cagctgctca 44280 ggaggctgag acaggagaat tgcttgaacc cgggaggtga aggttgcact gagctgagat 44340 cacaccattg cactccagcc tgggcgacag agtgagaatc catctcgaaa aaaaaaaaag 44400 tttgtcaacg gtttcactga atccagaata cttttctaaa atgtcaaccc tatagaatac 44460 attttataaa attatgaagg cctggtctgg tgtagtggct cacgcttgta atcccagcac 44520 tttgggcagc caaggcaggt ggatcgcttg aggctgggag tttgagacta gcctggccaa 44580 caaggcaaaa ccctgactct actaaaaaat acaaaaatta actgggcgtg gtggtgcaca 44640 cctgtaatcc cagctactca ggaggttgag acaggagaat cacttgaacc caggaggtgg 44700 aggttgcagt gagtggagat tgcgccattg cactctagcc tgggtgacag agcaagactc 44760 tatcttcaaa aaatagataa ataaataaaa attaaaacaa aataaaattc tgaaggcctt 44820 aggtcagaga attaccgagg gaatattcaa agttatacct ccaagtatct acaatgaaga 44880 tactttcatc agaaaaaagg agtttacggc caggccctgt ggttcatgcc tataatctca 44940 gcactttggg aagccaaggc tgaggcagga ggatcacttg aggccaggag ttcgagacca 45000 gcctgagcaa aaacgtgaga tcccatttct accaaaaata aaaatgtaag gtaggcatgc 45060 aactgtagtc ccagctactc gagaggctga ggcaagagga tcgcttaaac ccaggactcc 45120 agcctgagca acagagcgag accctgttta taaaaaaaaa agaaaaaaaa aaagaagaag 45180 aagaaggaga agaaaggaaa taaaatttaa gaaaaaaaaa aggacttaat aaggttgaat 45240 gaaggcaaga atattcttag ctctgtttaa gtcaagacct gagtagtagc tctacgtagc 45300 tgtatgtcga taatgttttt gagacagcac tactgataaa ttgttacata ataaactgtt 45360 atggctggat gcagtggctc atgcccataa tcccagcacc ttgggaggcc gaagtgagtg 45420 gatcacctga ggtcaggagt tcgagactag cctgatcaat atggtgaaat cccatttcta 45480 ctaaaaaaat aaaaattagc tgggcatggt ggcgcacctg taatcccagc tactcaggag 45540 gctggggcag gaggattgct tgaacccagg agacagaggt tgcagtgagc cgagattgcg 45600 ccattgcact ccagcctaga agacagagcg agactccatc tcaaataaat aaactgttaa 45660 attaagttta gcctaaagct acccccttac atattttaag ttcagtctaa aggtttccct 45720 gcacatagtg aactgtaacc taactggatg cgtaaacaga ctataaccta ctcttgggcc 45780 agtcactgag ttttggtcaa tcaaaggcag ccaactgttc aaaccaggtt aaaataaggc 45840 agatgctgag ctctaaccag tccagccatt tctgtacctt gcttccattt tctgtccatc 45900 actttccctt ttctgtccat aaatcttcca ccacgtggct gtgctggagc cactgtgaaa 45960 ctattctgtt tcaggggctg cccaattcat gaatcattcc ttgctcaatt aaactctgtt 46020 catttaattt gtctaatatt tttcttttaa tcaaagtaat ttggccgggc acagtggctc 46080 acgcctgtaa tcccaacact tcgggaggcc gaggtaggtg gatcacctga ggtcaagggt 46140 tcaagactag cctggccaac atggtgaaac cccgtctcta ctaaaagtac aaaaattagc 46200 cgggtgtggt ggcgggcgcc tgtaatccca gctactcggg aggctgaggg aggagaatcg 46260 cttgaacccg ggaggtggaa gttgcagtga gctgagattg tgccattgca ctccagcttg 46320 ggcgacaggg caagactctg tctcaaaaaa aaaaaaaaaa ttaattcaga gacctactca 46380 tgtgaagttg tattttttta ttctccatat tacaaaacag aacaattggc acagggatga 46440 agaaatactt tgcaaaacat ctagagaggt taaatgccat gagtctttaa aatgtaagac 46500 tgctttcacc tgagcaatct agtgtccatt tctagagcta gcttaaatgt ccgtgtaaat 46560 ccccgtaatt ggttgggata acaattacct atgttgtata acttgagtca aaaactacgt 46620 ttccactgcc tgccacccct atggatggtt ttctcttaag gtatcaaatt ttactgggaa 46680 agacctagat aaaatacagc gaaaatgagg cggggcgtcc tggcacatgc ctgtaatccc 46740 agcgctttgg gaggctgagt cagaaagatc tttgaattca ggagttcaag accagcctgg 46800 gcaatatagt gaaatcctgt ctttacaaaa aattaaaaat tagccaggca tgggggcatg 46860 ggcctgtagt cccagctact tgggttgggt gactgatgtg ggaggatcac ttgagcccag 46920 gaggttgagg ctgcagtgag ctctgaccat gcccctgcac tccagcctgg gtgacagagc 46980 aagacccagt ctcaaaaaga aaagaaaaag agtaatgtta ggtcaaggta gaacctacct 47040 tgactttctg ttactatgga agatattctg gggtatctct gagatccaag tattatggca 47100 cttaagtaat tcctatctat tgttctactt ggttcctcgg gagtaaaagt catattcaaa 47160 ccaaaaaggc tgtgggattt ccagaatttt aaaagcaata atagttaatg ttctcccatg 47220 ggagttactc cacattttta catatgttcc atatgttaac tcatttagac cttaccttta 47280 tgaggtaagt cctcttctta tccccacttt agaggtggga aaactgaggc acagaaagag 47340 taagttgctt gcctaaggcc ctgttactag caggtggtga aaccagcatt ccaacccggg 47400 agtctggcaa atgtgtgtga agagcacacg tttggaaatg acagtcatga ggacactgta 47460 agacttctgg aatgtttata atttcacctt tgcttgttat ttttcctgtc tgtttcccta 47520 gagtgagctg agtgaaaaaa gaaagaagaa agaaagaaga aagagaaaga gaaagaaagg 47580 agagaaaaag aaaaaagaaa agaaaaacag aaaaagggaa agaaagaaga aatgaaagaa 47640 agaaaagaaa agaagaaaga aaagaaagag agaaaggaag gaaggaaggt gggaggggag 47700 gaaaggaaga agaaagaaag aagggggaca gagggaggga atgaaggagg gagagaggga 47760 gggaaggagg aagaaataaa aagatgagga tctgtatgct tgaggggtgg aggtgggggg 47820 cttgggtggg agtgtgggat gggcagaaag ctggagggag ccctggaccg actgcattcc 47880 acagaggatt gtgggtgcaa cgtaggtggc agattgagaa aagcaaacaa acaagctcag 47940 cctttggagc ttcggggaag aaaaaaagct gagcagtgaa tgctggcttc ccacggagaa 48000 ggcaggctgc ttcgccagct cacatccttc cgcgcaccca cttcctcttt ccggaggtca 48060 ctttagattg ctttatggca ggatctccag gtcacaggaa tgttatgttt cgactggggt 48120 ttccccctcc cctgggatgc ctgggccagc tccccaaggg ctagtctctg tcccaggccc 48180 cacactccca tagcactcag caaaagccta gagagagcac cgcaaaatgc caaacgcaac 48240 aggaccgcgt aggaagaaga cgcttggaat gacagggaca ctagaactgc ccatggtcgt 48300 ggtctcaaat ttttgttcca tggtctgaaa tactaaaagt tcttaaacag ctacttgatt 48360 tcatactatt gttttgaaga aaacagtgtt tgtttgttgt tttgtttgtt tgtttgtttg 48420 agacagagtt ttgctcttgt tgccgagttt ggtccatgtt ggtcaggctg gtctcgaact 48480 cctgacatca ggtaatccac ccacctctgc ctcccaaagt gctgggatta caggaaaaca 48540 gttgtttctt taaaacaatt atataggctg ggcacggtag ctcatgcctg taatcccagc 48600 actttgggag gctgaggtgg gtgaattacc tgaggtcagc agttcgagac cagcctggcc 48660 aacatggtga acctccgtct ctactaaaaa tgcaaaaaat tagccgggcg tggtggtgca 48720 ttcctgtaat accaggtact caggaggctg aggcaggaga atcacttgaa cccaggaggt 48780 ggaggttgca gtgagctgag atggcaccac tgcactccag cctgggcaac aagagcaaaa 48840 ctccatctca caatctcaaa aaaataaaat aaaataaaat aaaataaatg gttatataag 48900 ctaccttatt gatgcagtta caaatgagcc gctgaaacat ataaatttta aagaacaagc 48960 cacatatctt tcatcaccca cagcttcacc aactaaaggt gtatgtagta cttttgtgga 49020 aggcatttcc acatgctttg agggaccttg aaatactgct atgattacat gatttttcta 49080 aaaccagact actcctacat tacaagaatt gaaaagttca gagtaaatat ttgtaagacc 49140 tagaaaagat gatgttcttt aaaaaaaacg atgcccatct ttgtagcgaa aagaaagaga 49200 gatcagactg ttactgtgtc tatgtagaaa cagaagacat aagagactcc attttgaaaa 49260 agacctgtac tttaaacaat tgctttgctg agatgttgtt aatttgtagc tttgccccag 49320 ccactttgac ccaactactt tgacccaacc tggagctcac aaaaatatat gttgtatgaa 49380 atcaaggttt aagggatcta gggctgtgca ggacgtgcct tgttaacaaa atgtttgcaa 49440 gcagtatact tggtaaaagt catcgccatt ctctagtctc aataaaccag gggcacaagg 49500 cactgtggaa agccgcaggg acctctgccc tggaaagcgg ggtgttgtcc aaggtttctc 49560 cccatgtggt agtctgaaat atggcctcgt gggatgagaa agacctgacc atcccccagc 49620 ccaacacctg taaagggtct gtgccgaggt ggattagtca aagaggaaag cctcttgcag 49680 ttgagataga ggaaggccac tgtctcctgc ctgcccctgg gaactgaatg tcttggtata 49740 aaacccgatt gtacatttgt tcaattctga gataggagaa aaaccgccct atggcgggag 49800 gcgagacatg tttgcagcaa tgctgccttg ttattcttta ctccaccgag atgtttgggt 49860 ggagagaaac ataaatctgg cttacgtgca cgtccagtca tagtaacttc ccttgaactt 49920 aattatgacg tagattctgt tgctcacatg ttcgttgctg accttctcct tattatcacc 49980 ctgctctcct actacattcc tttttgctga aataacgaag ataataatca ataaaaactg 50040 agggaactca gagatggtgc cggtgcaggt ccttggtatg ctgagcgccg gttccctggg 50100 cccactgttg tttctctata ctttgtctct gtgttttatt tattttctca gtctctcgtc 50160 ccacctgact agaaatatcc acaggtgtgg aggggcaggc caccccttca catcttgtct 50220 ccacttcctt gattaaaaaa aagaaaagaa aaaaaaattt gccgaagttg gattcattca 50280 cagaattcta cacattaaaa atgttgcagg tcgggtgtgg tggcagctcc caaagctgcc 50340 tataatccca gcgctttggg aggcttgagc ccaggaggtc aaggctgcag tgaactgaga 50400 tcgcaccact gcactccagc ctgggcgaca gagcaagacc ctgtctcaaa gaaaaaaaaa 50460 aaaacagaaa aaaataacgt tacagaaaaa gtacaatatt tttaatatat atatatatat 50520 tttttttttc tgagacagag tgttgctctg tcacccaggc cggagagcta tggctcgatc 50580 tcagctcact gcaacctcca cctcccgggt tcaagcgatt ctcctgcctc agcctcccga 50640 gtagctggga ttacaggcac ccaccaccac gcctggctaa tttttgtatt tttagtagag 50700 acggggtttc cccatgttgg ccaggctggc ctcgaactcc tgactttatg atccgcctgc 50760 cttggcctcc caaagtgttg ggattacagg tgtgagccac catgcccagc caaaagtaca 50820 atatttttaa tgacatataa agatgttcat tctttgtggt tgccctgggt gagagggact 50880 attgatactc aatagtgttt cttttgtttc tacattgttt ctatagtgaa aatacgcatt 50940 ggctttgtat taaaaaatgt atagtaaaaa tggttttatt aaaaatagca aataactaca 51000 aaaactccat tgcaatggaa agcagccctt ggattttcta gttgaatgaa acgagtaatt 51060 tatccaatgt tagaaatgtc taaaggctcg ctcaggtttc atgagcagaa caggaattgt 51120 atatccaatt aaatgtgaaa ttgcaatgcc tggtgcggtg gcttatgcct gtaatcccag 51180 cactttggga agccgaggca ggggatcgct tgagcccagg agttcgagac caccatgggt 51240 aacatgggga ggccccatct ctacaaaaaa taaaaatcgt tagccgggca ggttggtgca 51300 tgcttgtgtt cccagctact tgggaggctg aggtggaagg atcctctgag cccaggagga 51360 tgaggctgca gtgagacatg atcgatgcac tccagcctgg atgacagagt gagaccctgt 51420 ctcaaaaaaa aaaaaaaaga aaagaaagta caatcgcaat taaatgtctt tgcgttggtg 51480 gctcctgacc aaattcccta agcaagcagt atgttaatga gcagaggggc cacagctcac 51540 cttgctcaat taaaggcagg agcaggccgg gcgtggtggc tcacgcctgt aatcccagca 51600 ctttgggagg ccaaggtggg cggatcacga ggtcaagaga tcgagaccat cctggccaac 51660 atggtgaaac cctgtctgta ctaaaaatac gaaaattaac tgggcatgtg gcatgagcct 51720 gtaatcccag ctactcggga ggctgaggca gaagaattgc ttgaacccgg gaggtggagg 51780 ttgcagtgag ccgagattgc accactgccc tccagcctgg tgacagagcg agacttcatc 51840 ttaaaaaaaa aaaaaaaaaa ggcaggagca agtatgggcc agacagaaat caaggtgtaa 51900 attgggcaga tcctcaggcc cagtgctgaa ttttggtttg atgaaataaa acattacatt 51960 tcaaggttgg cagagaggaa tgaaggtgga agaggaatct agggccattt agggaagcca 52020 tgaagcctcc tgcccacact agtgggtaga gtggagccag gcgttttgct agggcttgct 52080 atatctcttg gcagggtgct ctgctgccaa agccaagaat tctaaattag attaaatagc 52140 cagaaagaat gttaaacatt tggacatgat atcctccctc acagattagc tagagtgtag 52200 ttctgctgtg ctagatactt aaataaatac ctccctagct gtgaagcctg cttatcacag 52260 tactatattt taggatgagg tcattatttt cctatgcata cacatgcatt gtataatctt 52320 gccaatgtag gtcagcccaa aagaagtgac aaatgtgtag aacacacatt ggactagctt 52380 gggacaaaat tagtatacct aaagatgaca gatttcttaa ctaattttat gagccatgca 52440 gctttgtatt ctagcagaga cagacattag gaatcttata aaatcaaaaa ttttaatttt 52500 tgcctgaata gctccaaagg gctaagatct caagcaaatg cgtgtaggtt ttgtttttgt 52560 ggttgttgtt gtttttagag acagggtctt gctctgtcac ccatgctgaa gtgcagcggt 52620 gcagtcctag ctcactgcag ccttgacctc tcaggcttaa gtgatcctcc tgccttagcc 52680 tcccgagtag ctgggactac aggcgcatgc caccaccccg agtaattttt tatttttatt 52740 tttacttttg tagagacagg ggtctcaata tgttgctcag gctagtatct tttttctttt 52800 tgagacagtc tcgctcaatt gcccaggctg gagtgcagtg gtgccatctc ggctcactgc 52860 aagctccgcc tcccgggttc acgccattct cctgcctcag cctcccgagt agctgggact 52920 acaggcgccc gccaccatgc ccagctaatt ttttttgtat ttttagtaga gacggggttt 52980 caccgtgtta gccaggatgg tctcgatctt ctgacctcgt gatccacccg cctcagcctc 53040 ccaaagtgct gagattacag gcgtgagccc tcgcgcccgg cccagtcttg taacttaact 53100 ttaaagctac ttattcccaa atgaagatgg gatggtacac agattttaag tattagctgg 53160 tttggagctt ctgtctttta aagcaacatt ttactttgcc acagggtggt ggggcggggg 53220 ccatcctaga aagaagagtg tgagtttcat gggatagggt ctggggaggt ggctggagga 53280 gtttaggttc ttttgatatc tgtggctaca cagacagata accaaggaaa atgtccaaac 53340 agtgaaatta agtgctcact gcactaacac agagaaggac cctgatgtct ggccgcaggc 53400 ctttgttctc attggcttca aagaacttct tgatgtctac cttaatttca ttattattta 53460 cccaggagtc attcaggagc aggttgttca attgccatgt agttatgtgg ttttgagtga 53520 gtttcttaat actgagttct aatttgattg tgctgtggtc tgagacactg tttcgatttc 53580 agttcttttg catttgctga ggaatgtttc atttccaatt atgtggtcga ttttagagta 53640 agtgccacgt gacgctgaga agaatacata ttctgttgat ttcggggggg agagttctgt 53700 agatatctat taggtccact tgatccagag ctgagctcaa gtcttgaata tccttattca 53760 ttttctgtct cgttaatctg tctaatattg acagtggggt attcaagtct cccactatta 53820 ttgtgtggaa gtttaagtct ctgtgtaggt ctctaaaaac ttgttttatg aatctgggtg 53880 ctcctgtatt gggtgcatat gtatttagga gagttagctc ttcttgttga attgctccct 53940 ttaccattat gtaatgccca tctttgtctt ttttgatctt tgttgggata aaattacatt 54000 ttatgtcccc cttcctatag tttgtcactg agggttggca gaagttgaaa ggaagaagac 54060 atttgggtgt ttggtttggg gttatattag gttataaggt tcattgcctc cacctctttc 54120 aaaacattta gtttctaaat gaatccagct ttaaatgact gcaggagtgc ccatgcacaa 54180 ttttgtttct caaatctttg ggatttttcc ttgaagaata ttcacaggga atggggctgt 54240 cttgcttcat agttactctt ttgtatacat gatctcaaga atcgcctgat cactgctaga 54300 gttaaaccaa tacactaact gcctgaagtg ctgaaaagtc aaatgggggc ttagaacctc 54360 actccagatc ctacacaagc tgatggttct gttcccagaa acaacccagc ttcctcatca 54420 tctatggcca gtgccttgta gcggagctgg agatcaccct ttagtgggct cttcagctgg 54480 atctagaaat caaattgaca ccaggcagat taacaagaga aaagtataca gattttattg 54540 cttttatatg tacttgggaa tctgcacaag ggcaaagtcc gaagaggtgg ccaaagcaag 54600 gtgcttttat acatttttag aaaaagagcc aaaaaattgg agaagaaatg ataggacaaa 54660 gaaaatctag ccaggcagta aattttctag gagaatcact aggacatata tgaggaaggg 54720 tgtgtaaaac aggtgaaaga taagggctag ttcattaaac atgtttactc tggtccattg 54780 tagcctctac gataaggagt attttctcgc tctggtgtgg acagggcacg cctcccagag 54840 caacctttat cacttactgc atgcaggaag agacaggtca gcccgccctt cctgaaacta 54900 caatttcttc agtgttttca actcaaaata atcaataccc cccatctggc atatctgggg 54960 atggcacgtc ctttactcct tcaggctctt ctccctgaag gtcctttgca tagttgggaa 55020 tctccaccag gaggggtagc tctttggtct aaacccatgg tggcagagtt tcgacaatat 55080 tcccaactta aatgtttctg attctgagtg gtggttagat ccctttgtac acccctgtcc 55140 ccagtgccta cagaatgggc atgttaataa gtgttggctg aacattcaat gatggataag 55200 gaagaatagg aggcaagaga gacggtggtc tccagtgcca agccccagtg ctaactgggg 55260 tgattttttt tcatgactca ttttcctaaa atcaccctca agggtcctac aaaactcttc 55320 ccaacagcta aatcacagac taatctggcc catcgacgtc ttccctgatt atactaattt 55380 ttttgtgttt ttttttttga gatggagtct tgctctgtca cccaggttgg agtgcaatgg 55440 cactatctca gctcacagca acttccacct cctgggttca agcgattctc ctgcctcagc 55500 ctcctgagta gctgggactg ccagcatgcg ccaccatgcc cggctaattt tttttttttt 55560 ttttttagta gagatgagat ggggtttcac catgttggcc agggtggtct tgaactcctg 55620 accgcaagtg atccgcttgc ctcggcctcc caaagtgctg ggattacagg tgtgagccac 55680 tgcgcccgac catatattaa tggtttttga tgaatttgtt ccatagatta aaatcttgtg 55740 ccccatcgcg tgtggggctc catcgcatgt ggggcacagg gttcctgagt gtttgtggct 55800 gtcaaaccaa gatgatttct tgcttaatca agcagatttg aaagttcatc tctgctacca 55860 ggaagcactt gctcaactca gaagacaatg tcctatcagt ctttcactat cacgcatctg 55920 ttcttcaaga tccgtcaaat tagctccagt gaaacggagg ctaaagtgaa actttttctc 55980 ttatatagat ttttattcat aactagggaa aaattaggca cccacagaaa aataataacc 56040 taaaaaaatt aggctgaacg taagaaaaat ttgtgatgaa ataaacattt caatcaacag 56100 aaaatatttt tctgactttt tatgtgccac cattagttac atcattgaga aaacaatatt 56160 tgtattaaaa aaagagctgg tgaaaatctg gcaattggtc gggcatagtg gctcgtgcct 56220 gtaatcccag cactttggaa ggccgaggca ggcggatcac ttgaggtcag gagtttgagg 56280 ccagcctgac ccacgtggtg caaccccctc tcaactaaaa atacaaaaat tagctgggcg 56340 tggtggcagg cgcatgtaat cccagctact agggaggtta aggcaggaga attgcttgaa 56400 tctgggagat agaggttgca gtgagccgag actgagccac tgccttccag cctggtgaca 56460 gagcaagact tcatctctct ctcttttact ttttttaaag acttcttctc aaaaataaaa 56520 agaaagaaag aaaatctggc aatccagtaa aaactggcca ctatggcatg catgtgctat 56580 gcataaacgt aaattgatgc ataaacttaa ttttagaact ggaaggaaat ctggagttct 56640 ttaggagcca ggttttacac atgcagaaac ctaacagctt cagtttcgat tcgataaaat 56700 ttgactaact aaacttaaga taagcatagt tacgcattag agtattaact ctcaaacttt 56760 taaaaaagaa ttcttccttt gcttgttaat tttctttctt tctttttttt ttttttgaga 56820 tagggtcttg ctgtcgtcca ggccgaagtg cagtgacgtc atcatagttc actgcagcct 56880 ctacctcccc ggctcaagta atcctcctgc ctcagccttc tgagtatctg ggactacagg 56940 catgagccac catgcccagc cttttctttt cttttctttc ttttctttct ctctctgtct 57000 ctctttcttt cttttttcct ccctttcttc tttttttttg atggagtctt gcactatcgc 57060 caggctggaa tgcagtggtg cgatcttggc tcactgcaac ctccgcctcc gggttcaagc 57120 aattctcctg cctcagcctc ctgagtagct gagactacag gtgtgtgcca ccacgccagc 57180 taattttttt atttttctag agacggggtt ttaccatgtt ggccaggatg gtcttgatct 57240 cctgacctca ttctccacct gcctaggcct cccgaagtgc tgggattaca ggcatgagcc 57300 accgtacctg gccctttctt tctttttatc aagacaacaa catgtcttta tagtgctccc 57360 aaggctaaag tataccttac gtctatgtaa acactcaacc tgagctttgc aatggcccat 57420 gttggcagta gtgcaaacaa aaacaattat gaaacccatt ttcctttgac aaagagaaat 57480 aagtggcaag aattggttct ttctcttagt atgggtctct gaaaagaacc agatcagtca 57540 aaaggggaat atttttctga agggataggt ttggcctagt ggcttctacc tcttttagat 57600 gactgctgtt tctcgtttta atgttaaata gacactaata ggagaaatca cattaattca 57660 gtcaacaaac atttactgag cacttcctgt agtcaggccc tctgttaact tctgggaata 57720 caatgacaac tctgacaatc ccaacccaag gagccaacaa gtccgggaat agagacagac 57780 aagaaaacag acaattacaa ctctaccgtt agaataaagg tacattgaga acttgcaaca 57840 aatattcctc atcccttatc ttaattattc ataacatgtt taccaccaat aagaatagca 57900 ataacaataa atgcccaact cagacagcaa tgtccattta ccctgtgttt acacagcata 57960 atacaagcaa gctgtggaca gagattctct tgtttagtcc tcacaactct gcaaggtggg 58020 ttttattact ctccatttct agataaagga tctcacctaa tattacatgg gccagtggtc 58080 ttccagttgg ggtatgcaca accctagggg taggtgagga ccctgcctgg ggtcttcagg 58140 tggggaccat caacctccat ttgtactctt ttctgaacat tggtctgaga cagaaagtcc 58200 ctgcaattaa ggcattaagc tggctctttt tctatttctc atttcataat tgcccttctc 58260 ctgctttacc aaaatctttc accccccatc atatatatat atccccatac atattctata 58320 tatacatacc ctacatatgc atgcacacac atcatatata tgtatgcata tatgatatat 58380 acatatatgc tatgtaaaca tatatagtgt gtatatacat gtgtgtatgt gtatatgtgt 58440 gtatatgtgt atatatacac gtgtgtgtgt acatatatac atcatatatg tgtgtctata 58500 tatgtatata tgggtgtgtc tatatatgtg tatatgtacg caaatacgta tatgtgatgt 58560 atatatataa gatgtgtgtg tgtatatata tgtgtttgca tgtgtgtgtg tatatatata 58620 tagtatatac atattttttg agacagcatc tcactctgtc gcccaggctg aagttcggtg 58680 gctgatgaca gctcactgca cctcccggct caagtgattc ttccacctca gcttcctgaa 58740 tggctgggac tagaggcgtg tgtcaccaca cccagttagt tattttattt ttcgtagaga 58800 tggtggtctc actgtattgc ccaggctggt ctcgaactcc tgggctcaag cgcttttcca 58860 cctcgacttc ccaaattgct gggattacag gtgtgagcca ctgcaccggc ccatccttta 58920 ttttaatatt atgcagtgcc ctgagacata taaaaaaccc accttcccaa gtaaaggaaa 58980 ttcaagctga tgcctgcaga gccttcttta acaaaggctc tgaaataccc tctctcatta 59040 aaatgatact ttccaataaa attttgttta acaatgattt acaaaatgat aaaatttatt 59100 tattttgatt gtgtatggat catggtaaca ataaaaagac ttgtaaaaat aactaaattg 59160 aaagaatctt gaacatttag agccttaaga ctgtaggaat tgaagaccac agaattatta 59220 atttatatta atatttttgt tgcagagaca taatgaatga tcaacgaaag gcttttaagc 59280 gttaaaaata tattacacta gataaaatta tttgcgggaa tgggatggaa atacattttc 59340 aagagagaaa ggagcaatgt aaaatgaaga tgtaaaatcc ttctgctggt tgtccttggg 59400 gttttctttt aaagaaaagc ttggcagtgt ttttcttttt ttccattgga tgatggtgaa 59460 tatcaaatca ctttggtgct aatatttcat ttaatacatt aattttaaaa ttttctgtag 59520 aggtgggatc tcactatgtt gtccaggctg gtttcaaact cctggcctca agcaatctcc 59580 ctgcctcagc ctcccacagt gctgggatta caggtgtgag ccactgcatc cggccccatt 59640 taatacattt aaaagagtgg tgtaacaatt tttatttaaa atgtcatatt tacaatattc 59700 tagaatgtat atcttttcaa ctcattaaac ctaaacatcc ttgtaaaaag tgtgaaaagt 59760 tatatagttt ttcaaaattc gattagcagt tacataagca taaatgttta aagtatgtat 59820 ggtacagcca ggcttcagtt ccctgtctta aacacaaaga tccatatcaa ttccagatac 59880 tgcaatggtt tgctgttttt cctgcttccc ccatctccaa ataaactaaa gcatcaacat 59940 gcctcacctc acataaccct aagttttcag cagttggcag ttacacctgg aaaccatttt 60000 tctaaaataa acaacaactg tttgcttacg gatcaaaatg caaaggacca taacatttag 60060 cctcaccttc ctactacaga tcgagtttaa aagtgccatg gtatagctaa attatgaaga 60120 aagatatgaa tataactgca aaagtggaag gagatttggg ataattcttg cccattttgt 60180 taggccaaat gcatctttgt gcaaattaga aaaaggtggt cttcatccct tcactcctat 60240 ccttttgggg gtggaggggc agtggctaaa gtacagacta ggtttcagct accacatcct 60300 ccttcagtta gctgccctcg gcgtgacaga aacatgtgca aacagccctg tgcctttgtc 60360 ttatgttcca gccagccaag aaaaatagtt gtaaaagagc agctgctgtt tggggtaatg 60420 accttggacc ctccccaatt tgttccaagc ctgtttttgt attcattttt cccacattta 60480 tgttcctgga tggaagcttc catatctgct cttggcccta tttgaaattc cccagatttc 60540 cttcctggct cctggccttt ggtttttcat gtggctcctg atcccacacg ctccctgaat 60600 ttggattctc ctgtcatttc aggtgcgagg tttcccacta cagcctcttg ggcctcacct 60660 ccaatacctc tttcccatca gaacagcccg gaccttcccc tatggtagag cagagacaga 60720 atttaaatga attctcaaga agtgcttgga ctcatatcta gcaaaattac atggcattta 60780 acctttgaca caaaaaatgc agcttctagg aatctatcta aagatacact gtggcaaata 60840 tacaaaaaga agcattattt atcaagcact atttcctaat aaaataattc ttaggtcagg 60900 cgcaatggct caggcctgta atcccagcac cctgggaggc tgaggaaggc agatggcttg 60960 agctctggag ttcaggacca gcgtgggtaa catgacaaaa ccccatctct aacaaaaata 61020 caaaaattag ccgggcatgg tggcatgcac ctgtagtccc agctactcga gaggctgagg 61080 tgggaggatc gcttgagcct gggaggcaca ggttgcagtg agccaagatc gcaccactgc 61140 actccagcct gggtgacaga gtgagaccct gtcaaagaaa gagagagaga gagagaaagg 61200 aaagaaagga agaaaggaag aaagagaaag gaaagagaaa gaaagagaaa aagaaagaaa 61260 gaaggaaaga aagaaagaaa aaagaaagaa aaagaaagag aaaggagaaa aagaattctt 61320 actaataaat gcaggagaaa tgatagaatt gaaatatcac cattttcaat tcctaatgaa 61380 ataacgtatc taggcaatga ccatcaatag ctagatgcta aaatcatctg atcaaacact 61440 gatgggaact tcgtaacaga tggatcaggc taacaacatc tgaaaccact aactggtttt 61500 gatgtcataa aaagaaaaac aaccagatat tttctgtctc ctgatgagtt gcaattggag 61560 ctacatatca cctgtaaagt cttctggcca aaaaattaag cccagccgga ccttattaaa 61620 cctttaaatc taacaattag ttttgaagct tttacagatt aaatgaagtc tgagatttgc 61680 ttcaaaatga accagtggtg gggaggaagt gggtgaggtg taggtgaaac aagattggcc 61740 acgtcgataa ttgctggagc tgggcgatga aagcacaggt atttatcaca ccatctctct 61800 acttttgtgt gtttttttgt ttgtttttgg ttttggtttt aaggagcaga gagtctaata 61860 ggcaagaaag aaaagagaag gctgaaggaa gacgctcccc cgtacagaga cagagggagg 61920 gggctccaaa gccgaaagag gaggtcctct tgtgtatgtt ttaaaatact cccagataaa 61980 atatttttgg aagagtactt ggttggattc aacagctttt ttttaattta aaaaaatcac 62040 ctcaattttt ttgcttgctc taacgtgcca tagaaattcc tgaggtttta cttgttgctt 62100 tacaatgaac tgtgtaaaca caagctggaa gagatcagct atgcgctgga agggttggtt 62160 aaatattgag actgccttgc tgagggaagc cttttaatga atctcagtaa ttttgcaaga 62220 gaaaagataa caatgaacac tacattaaac atcattcttt tgcactttgc taaatatgtg 62280 tatgtaaatt actgtctgac tgttactgga tatatacagc atatacatat gcactttttt 62340 tactgttttt tttttttttt tttttttttt tttttttaca gagcttgctc tgtcacctag 62400 gctggagggc agtggcgcag tctcagctca ctgcaatctc cgcctcccag gttcaagcga 62460 ttctcctgtc tcagcctcca aagtagctgg gactacaggc gcctgccacc gctcctggct 62520 aatttttgta tttttagtag agacagggtt tcactatgtt gtccaggctg gtctcgaact 62580 cctgacctcg tgatccatct gcctcggcct cccaaagtgc tgggattaca ggcataagcc 62640 accgcgcccg gcccatgtat actatttata catttttagt atcattttgt ctttacattt 62700 tacataattt cagatacatt ttcctcatat caaataattc agcatttttt agtactaaca 62760 tcatagtctg taagccattc aaaaaatgta tttcacaaaa taggctatct catcctttga 62820 gctattgaga tgaattaatt tatactcctc ctaagatccc tctcgtcact aagattcttt 62880 tattttatga caaaaccata gttctagaag cttgtttctc ccacctgaaa agactggatt 62940 tgggacatga tcctgtagaa cttcggaggt aagcctggtg aatcagatca tagggggtct 63000 ggagggtgaa aaaaaagggt ttggtgctca tggatggggc tagtattggg gtgtagggga 63060 gattaggtca aagcaagagg attcaaagga gaaatgaatt cctttagatt ggggaagata 63120 atcggaagag gtaaaagaca ccgtccatga cacttcctgg ggaagcagat gtatgtataa 63180 ggatgtgagt attgtggttt tgtaaagaat gcattcctga agatgttgca taatttaaaa 63240 cctacatatt ttgattaatt ttctcatgag aatagcaggg tatgtgttct cggcgctcac 63300 aaatgtataa tccattgtgg caaatttttg ctttcacata ttttttttta tcattattgt 63360 cacaggttct gtgacggagt tctggtttct aaattcacag cataccaagg cagttcttta 63420 aagttcttga tactctttta tcatatctaa cttgtattcc aaaattattg agttggagca 63480 cattttccca gcacttagca ccgctatttc atggatggtt ggagaggggg tccaaaaatt 63540 ttacaattat gttaacaaaa gtaacacagc aacaaaacaa taagcaaaat cactgccaga 63600 gtattcctta gcttgaaaca atacggttca catcgataga atatggcatc tatttctgtt 63660 taatcagtta accctgctaa gtagcaagag cttacaattc atgtctaaaa tcatgatttt 63720 tttactagtt ttttaaaaaa tgtgggctct atatatataa tttaacattt tgcttgtaag 63780 acttaatttt gcctgggtat ggtggctcat gcctgtaatc ctagcacttc aggaggttga 63840 ggcaggagga ctgcttgaac ccaggagttc aagaccagcc tgggcaacac agtgggaccc 63900 catcactacc aaaaaaaaaa aaaattagtc aggcatggtg gtgtgcactt gtagtcccag 63960 ctacttggga ggctgaggtg ggaggatcac ttgagcccag gaggtcactg ctgcagtgag 64020 ccattattgt accacaacac tccagcctgg gtaatagagt gagactctgt ctcaaaaaaa 64080 aaaaaagact atttctaaat gtgtggctat attataccat ataaatgtgg cttcttgggc 64140 aaggaaagag gacaatatag atgaaaaaga aattgatcct accagaagtg atccttttat 64200 ctgcataact ctcaggcagt tgtggcaaat aattggcaat atctattgtt ctgaaactgg 64260 ttttcgcaac ttttattggg aacaccatcc cctctcctgc atgatcagtt tctcctctcc 64320 acggatcatt cacatgagta aagtcagtag cgtgctggta aatgtttaag atcttgttct 64380 ttgggggaaa aagttcctaa gttctagcag ttgccctgga taacttcaag gtatcaacat 64440 ggaagttatg tacaaaaatg gctgtcacaa gccagtatga gctaacacca acatactacc 64500 cagtgttctt caaacttcag ctcacagccc attagtgggg cttgcaaaca ttttagtgga 64560 ctataagcag cattttttta aaatgaaaaa gtagattgtt ttacacataa caggagtatt 64620 gttttgtaca attttttttt tttttttttt tttttagaca cagtttcact ctgttgccca 64680 ggctggagta cagtggtaca atctcagctc actgcaacct cggccttctg ggttcaagcg 64740 attctcatgc ctcagcctcc cgagtagctg aaattacagg catgcgccac aaggcccagc 64800 taattattgt atttttagta gaaacagggt ttcaccatat tggccaggct ggtctcaaac 64860 tcctgacctc aagtgatcca cctgcctcgg cttcccaaag tgctgggatt acaggagtga 64920 gccaacgtgc ctggtcaaaa tttttgtttt cgtaatttta agtatgtgtg tagtaagtct 64980 caatggaaat gtaattctta tggcaggtca cttgaaaaaa aagaagtcta aaagtcacca 65040 atgtagtatc ttctctttaa aaaaaaaaaa aaacaacagg agaaaacctg aatctgccct 65100 ttgctccact ccttcctcag ctataatgct gcttctccat tcctcctcac agcaaacctt 65160 tctgaaatct ttatagtcat ggtttccacc agttcttcac ctcccatttc tcaacacact 65220 tcagagtcag agtcagcaat gacatccatg ttcctaagcc cattgcttac ttccgtcctc 65280 cttggcctct cagcacactt ggcacacagg ctgtttctct ttctttggca tctgtgacac 65340 cactctcagc aaattcccct ggttgtccct tctcagtctc atttattggc gttgtcttat 65400 ctccccaggg ctgtccgagg tgattttctc ccactactct cctaggtggt gccatccaat 65460 ctcatgatgt catatcattc ttccctcatg cttcagccat actggttggt ggcctttgtt 65520 tcctgaacac atttaatgca ttctcaagac cctcagggac tttgcagcag ctgctcgcta 65580 aggctggaat gctcttcccc accatcttca tatggctgtt tcctttcttt cactcaccag 65640 cagcttaaac tttgactcct ctgagagact ttccttgtca cccaactaag gttgccactc 65700 aggtgctccc aatttaatct tctctaaaac acatcactgt atgtgtcctc aactagagta 65760 taagcttcct cgaaacaaga acaatcaaaa ctccttgccc tcatggagtt tatagtctta 65820 tgatgggtga agtaacataa aataaaaagg caccttatat agtatattag catgacaaat 65880 gttagccaga aataaagcaa ggaagagttg ctagggagtg tgtatgagtg tgttttggga 65940 gagtgtttgc aattttaaat attggtggtc aggaagggcc ccactgagaa ctgacatttg 66000 agtagacttg aaaagggaaa aggaaatatt gagtaaagat tttaggatgg gagtgtgaca 66060 ggcctgctag gagaatagca aagtcgctgt ggctgctgca gaaaaagtga gaaggaaagt 66120 agtaggagat gaaatcacag tgtgtgagga ttcgggcaga tcaggaagtg ctcgtgtaag 66180 aactggatct ttactcaaag aatgagcaaa aattagtaga cggttggccg gatgcagtgg 66240 ctcacacctg caatcccagg ttacaggagg ccgaggcggg cggatcactt gaggtcagga 66300 gttcaagact agcctgggca acatggtgaa acctcatctc tactaaaaat acaaaactta 66360 gctgggcatg gtggcgcgca cctgtagtcc cagctactca ggaggctgag gcacatgaat 66420 cacttgaaca cggtaggcag aggctgcagt gagctgagaa tgtaacactg tactccactc 66480 caacctgggc aacagagtga gactgcctca aaaaaaaaaa aaaagtaggt tttagtaagg 66540 gattaacatg atctgaatta tgttttgtca tgacttctct ggctgttgtg ttgagactac 66600 attgcagagg ggcaagggca aatataggga gaccgattag gatactgcag taataatgta 66660 agagatgtgg gactctatct agaagggccc atgaggtcct ttgcatgcta gtattcttta 66720 ctgctgtgcc tggccatgat aggcattcag tgaatatttg cttatttaaa ataacacact 66780 gggctaattg aacaacagtg ccaaatgagg gagatatttc taggaataag ttcttaggat 66840 ttatgaacat tttaatccag attttctttg ttaactctgc tctctggccc tttcactcag 66900 ccccgtttgc acctaaatat gacttacaaa agaaacacag catttatgtg tacttatttc 66960 aacttacttt agctttgtaa agaagtacaa ggttgactca gggcccagct tggtgtctca 67020 tgcctgtaat ttcagcattg tgggaggcca aggcaggaag atattgtgag cccaggagtt 67080 tgagaccagc cygggcaaca cagtggaccc tgtctctaca aaaaaaaatt tttaaattag 67140 ctgggcatag tggtgtgcgt ctatagtccc agctactcct ggggctgagg tgggaggatc 67200 acttgaggga aaccctgtct caaagtggcg gggctggggg gagactcagg cagaattgtg 67260 aagatattca attgctcctg actttatcaa taatctaaca tttcaaccta acattgatat 67320 ctattttatg caaagcatta cactatgcac tggagactgt ataagacaag ttccttttct 67380 caaactacag tcgagttgga tagataaaac acacaacaca taccaaaaga cagctataaa 67440 tccaaggcag tgtatgtcaa gggtaaattc acctattcag attggatctt gagaagtgca 67500 tcaggcttgg aaaatgggta aggaggagag aaaagcaaca gtgaatcaga acatgagttc 67560 ccagttatgg gacttgtaat gaattcctca attaaaacaa aaaataatga aaacaaaagc 67620 cagggaggag aaagcccacg ttaatgacac taaaatatat ctttccaaac aaatgtggat 67680 aaaagccaag tagagaagat gagaactttg aggtccctaa cacaaaataa acagtaagca 67740 gccagccatt ccaagtggct gacatgactt tgtttaactt tatttgtatt tctggctggt 67800 gtgtttacag ccaataggtc aaactatcag tcagtgtagg gccctgagaa gtcgggtatt 67860 taagagcatc taataggcac agaattgtgc tccatactgc ttaaactgtt ccctaagtgt 67920 ccaatttgga gaaaacaccc acacgcagga taaccggcga gtgacgcgga gtggctgcga 67980 gtccaagtta tcactaacgg atggggagct tgggctgggc acagtccagc gtactgaacc 68040 cttcccccac cgtttcacct gcatacagag gtgtgtactg tcaaaaagca gcgcctccaa 68100 gtctcttctg gcactgtctg gacttggatc cgaggcagac gaggaagctg agaaaaccct 68160 ggcgttgacc ccgtggacct gggcgccccg ggaaggccag cgcttggtcc aggcaggcgg 68220 ggcctgtgcg gtgaccaccc tggtcctgaa aagtcccagc cccgagcgcc ctccctccta 68280 gacctggagg cctggaacag ccaggtggac gtcggcccac ctttcttttc tttccttccc 68340 attttcctac cacctcccac cccactccgc cttccgggca aaggcagcca gatccaccca 68400 ggacacattc tttgtcctta tccctctgtg ctcgtcccac agcaagccag tcgcggtcca 68460 aggctccaga ggctgtgcag gaggccgagc tgggtggcga tcagcggcgg gtccctgtcc 68520 aaaayccagc agagccgcca gggacgcccc agacacagaa ggcggggcgc ggggagggtg 68580 gggagaccac agcagtgagg cgcgcgagcc gggaagtgaa cgaggactga ctcctgtcgc 68640 ttcccgtagc cgcccacgga cgccagagcc gggaaccctg acggcactta gctgctgaca 68700 aacaacctgc tccgtggagc gcctgaaaca ccagtctttg ggtgagtcgc gcgacccccg 68760 gcctcgggtg gcggggcagt cgctagaggc gtggctgctc tgagggtctc gccagtggag 68820 gatggcattc ggatgtcacg gctcctaaat caccatttga tgggtgggac agtgtccagt 68880 ccaccccgac ccgccggtcc tcaccgcggc agagccgggg ctgggtggcg gggacgctgc 68940 ctctgcaggc gaggcgctcc ggggcataag ggattatcag gagtcgcggc ctttcttgga 69000 catccctggc tggggtcagg ctgtttgccc tggggtgtct cctcgctgca aacccacccc 69060 acctgggctg ctttctcacc tgttccctcc tagcctgagg ccgagcgcca cctccaagtg 69120 gaggaatctg gggaagtttc cttcccggaa tttgtagtga cagtggagtg acctccattg 69180 cgttccctgc ctctaacacg ctctttagga tgccgagtca tttgactgca gtgttaaaca 69240 ttgcaaagcg caagtcatgt gacttccttt gaccgtacgt gaaacttaag tgatggctgc 69300 ttgtgatgca tacgaagtgt tcatgctggc gggacctgtc cctggggata cttcgggggt 69360 tgcgtgattt aatgcaagca gatggcttaa attgggtcac tggcttgtta ttatacatgt 69420 gtatggcaac tcggcatcca ttctttttgc tcttgttctt acttcctgaa ttgagtcacg 69480 gagccagagt tttgaggttt tgactaacga attaagttaa tgacatgggg ctatatttag 69540 gtggtaaacc aagagggata cagttttttt tcttaataaa gaaaaagtga tagatttgat 69600 cggtgtgtat tgttggtgtg cagtataatg acagaattgc tggaagtaaa atacaggaag 69660 ctctggtttc atttcccctt tagttctgct taaagtcgag tttttcctgg agctattaaa 69720 tgtagtgtag tgtccatgag tgcttttatc ttaaaaaatg tggctgatgc tttccaacac 69780 tcccctgccc tgtgattatt atttttttaa gcaacagaga aaactgtatc ttaatagtat 69840 taaaagtatt ggatttttcc ctactttgat ttgtttaaat tggaggagga agagcaattc 69900 tttctattca caataataat agctaacata gcgcttactc tttcgctgtt ttattaactc 69960 aatcctcaga acaaaccaat gatgtgaata ctgtaattct cattttatgg aaatgaaaat 70020 ttaaatgaat acctctgata attgtacggg actgtttgat tagtatttac cattaattaa 70080 ttaaattttt tttttttttg agatggagtc tcgttctgtt gcccaggctg gagtgcagtg 70140 gcacaatctc ggctcacttc aacctccgtc tcccaggttc aagcaattgt cctgtctcag 70200 cctcctgagt agctgggact acaggtgcat gccactacgt ctggctaatt tttgtatttt 70260 tagtagggat gggatttcac catattggtc aggctggtct ccaactcctg aacctcaggt 70320 gatccacccg ccttggcctc ccaaagtgct gggattacag gtgtgagcca ccatgcctgg 70380 cctattttag tatttttaat aataaattcc atgttagaaa ttttctactg atgtattttt 70440 taagtcaata tttcctacac tcacaatcca aaattattta gtatatgagc acactggtaa 70500 gaatgggagg cagatcgttg attgtaataa tattctatta tttggtaaat atcagtaaca 70560 taatatataa tttaaatttt aaaataggat atgaagaaaa atgctacatg cttacttttc 70620 ttttcctcta tttttacttt acacagggcc agtgcctcag tttcaatcca ggtaaccttt 70680 aaatgaaact tgcctaaaat cttaggtcat acacagaaga gactccaatc gacaagaagc 70740 tggaaaagaa tgatgttgtc cttaaacaac ctacagaata tcatctataa cccggtaact 70800 gatttctata agataacttt ttacctatgc caggacagat ccaatagaat attaattatc 70860 cattgggaga cagggcaaga ataaaagcca gtgaacatat ttaaagcacc tactatgtaa 70920 tagagatggt ggtgggtgct gattacgaaa cagctcttgt cctctagtgg aggaagaagt 70980 cacaatgata atatgacgtg atgaaacagt gttatgaaca gggaacgtct gggtagagtg 71040 gagggaatgc caacttttgg tgatgggagg aggctcagct aatcataaat tgtagttttt 71100 aaaggaaaat ggatttctta ctctacaagt ttttcatttt cttttttaat tagagctgtc 71160 catgagaagt taatgtctcg atctttccct cagcctttca aatactgctt ggcccttgag 71220 cagggaaaat gtcaaaagcc aatggggaga tggagagtgt gaagtagtaa gggtctcgtg 71280 cagttcaggc aggtcctaga atccctgaat gactgtaatt gctggaaatt gccctgtaat 71340 cctgagcagt aaagagcttg ttttagtttt atgtggtggt gagaatcttt aggaatgtct 71400 agtttccacg tatctgaagc tgaatcctga atcgaggtct gaaaaaggac agccactttt 71460 ttagtaaacc gcctagaaga ttcttgggca aaaggaaggg tgagaatcct taaaatgagg 71520 ccctaaacca gttttgttag tgtgtgtggg ttcaagtttt tgtcatttac tttatagctg 71580 tatttccttt ttccctaagt tttaatgtca ttgtgtaaga atgaggtatc gctgctgtat 71640 caagcaaagt cagttttagg agaaatagcc tttcagtggt agtaagttta aaaaagatga 71700 cttcctgaag cggaagcttg tgagacattt aagatgactt tgcgcatgtt agagttaaaa 71760 acatcccaag gttgtaaact gatttcctgc aaagatctta acaacaacaa caacaacaac 71820 aaactaggct gcctgccacg ggtgtctgaa gtatcatctt ggctcaagct gggagaatgg 71880 ataaaggtta cactgttcat ttctgccctt cacacagaaa agaagataat tttataggta 71940 aaattcgtgc atatcttgat tctagcatac tgctgattcc tgtagtttct ggggtcagta 72000 ctctcaacta ttgaggtgga acaaaaataa gtagacttca tttcttgagg aaggggatct 72060 ggagaagtag ttctgcgcta gagcagaaaa tgccttcagt cttgtggcat gggctggatg 72120 ctgttctgag gataatgcat ttccaaggga gatatttttg gcaaatagct tttttttctt 72180 tcttttcaaa attctctgtt ttattatcag ttctcacaaa agagtcggaa aggttagagg 72240 tagactgaac tgaatggcaa aaacattttg cgctctcttt acgtttcact gctgtaaaat 72300 atttatagta taaagggcct gtattgcact gaatttctct catttgtagc tagttgccct 72360 ttcaatgttc caaaaaaaag gctgtaaata acttatttta tttattcaat taattttttt 72420 ttttaaattt tttgagatat agtttccctc tggtcaccca ggctggagtg aaatgatgca 72480 atctcggctt actgcaattt ctgcctcccg ggttcaagca attctagtgc ctcagcctcc 72540 tgagtagctg ggactgcagg cacgtgctac catgcccggc taatttttgt gtttttagta 72600 gatatggggt ttcacagtgt tggccagcct agtctcaaac tcctgacctc aggtgatgtg 72660 cccaccttga cctccaaaag tgctgggatt acaggcgtga ggcaccatgc ctggccaact 72720 tagttattta aagataatca attagtatat tttataagct agacttagga aaactgtttt 72780 cagctgggca tggtggctca cacctgtaat cccagcactt tgggaggccg aggcaggtgg 72840 atcacgaggt caggagttca agaccagcct ggccaagatg gcgaaactcc gtctctactt 72900 aaaaatacaa aacttagcca ggcgtgatgg cagcctcctg taatcccagc tactcgggag 72960 gctgaggcaa gagaatcact tgaacctggg aggcggaggt tgcagtgagc cgagatggtg 73020 ccactgcact ccagcctggg tgacagagcg agactccatc tcaaaaaaaa aaaaaccccc 73080 cccacacaca aaacctgttt tcttgaatca tggttgtttt gttactgata ggttcaataa 73140 gtaaatatat ttattgtctg ttgtattctt tattaggcat tataaacaca ccgccacttt 73200 ttaattttta tttcattaat gtttccaatt tttttttttt tttttttttt tttaagacag 73260 aggctcgctc tgtcatccag gctggagtgg agtggtgcag tcttacccca ctgcaacctc 73320 cacctcctgg gctcagcctt gtaaatagct gggactacag gcatgcacca ccatgcctgg 73380 ctaatttttg tatttttttt ggtaaagaca gagttttgcc atgtttctca gtctggtcaa 73440 gcactcctcc cgcctcggcc tcccaaagtg ttgggattac aggcatgagc caccatgcct 73500 ggcctatttc taatattttg gtccacattg gtgttagacc aactgtccac attaagtttt 73560 cttggaaaag atgaagtaaa tattgcaact ggcctatgta tttttttccc tatttagtat 73620 atttctttga ctagttcaac tgatagaatt ccaagactta aaaaagtcag gctctaaggc 73680 tgggtccaga ggctcatgcc tgtaattcca gcactgtggg aggccaaggc tagtggatca 73740 cttgagccca ggagttcaag accagcttga gcaacatagt gagaccttgt ctctctataa 73800 aaatacaaaa attaactggg gattgtggcg catgtctgta gtcccagcta tgaggaagag 73860 tgaggtggga ggattgcttg agcccaggag gttgaggctg cagtgagctg tgagtttgac 73920 actgtgcttc attctgggtg acagagcaag aaccatgttc aaaaataaaa ataaaaagtc 73980 agagtccggg tgctgcggct catgcctgta atcccagcac tttgggaagc cgaggcgaga 74040 ggatcacttg aggtcaggag ttcgagacca gcctgactaa cacagtgaaa ccccgtctct 74100 actaaaaata caaaaattag ccgggcatgg tggcggtggc ctgtaatccc agctacatgg 74160 gaggttgagg caggagaatc acttgaaccc gggaggtgga ggttgtaatg agccaagatt 74220 gcacaactgc actgcatcct gggcgacaga gtgatacttc atctcaaaaa aaaaaaaaga 74280 aaaaaaaagt taggcttcct tttctgtttt ttttttcttt tttcttctct tttttttttt 74340 ttttttaaga gatggaggct tgctctattg cccatgctgg agtgcagtgg tgcaatctcg 74400 gctcactgcc acctttgcct cctgggttct agcaattctc ctgcctcagc ctcccgagta 74460 gctgggacta caggcgcaca ccgccacgcc ccgctaattg ttcttttgta ttttagtaga 74520 gacggggttt caccatgttg gccagcctgg tctcgaactc ctgagctcag gcaatccgcc 74580 cgcctcggcc tcccaaagtg ctaggattat aggcgtgaac caccgtggct ggccacttac 74640 ttttctttct attgaatttg aatgaataat ttggaagaca gtatctttac ttcataccag 74700 gaatgctgcc agtgaaattt cttgtttggc agttcattat ctacctatat atttaatttt 74760 gctattgttt atagagttct taagatatga ttaaatgcta gctggttaag aaatcattta 74820 gaaatgaaac agaattggtt gttactccaa gttaataagt tgcttgtcaa cataaatcct 74880 acctggtacc cagttttctt aggaaccttg cttccatgtt tatccttttc tgcttagtat 74940 tctaagtact ccttttttac cttacaattt agtcttaaaa cacaacacag tcaagtcttt 75000 cttttgtaac ctgtgaggta ccttctagcc tttgtgctgt ttttcttctt tttttgctgc 75060 ctgccttcct gactgagagt ggatttcctc actaaggctc tgccctctga tttttcactc 75120 tcttttcttt tttggtttta ctagtgaaat tttgtcttta atgtctcttt cttttatgtc 75180 tttaccgatc actcataaat ttttttttcc atatgtatcc agttccaacc tttcacctaa 75240 tgtgaacccc caactctcag ttgctcagcc agcccttcaa gactaggagt tcaaaaccaa 75300 acttgcatct tccttcccaa accagctttc ctcttgcagt tttctgcagc aggatccttc 75360 tgctgtttaa cttttgcctc ctcccttgtt tcctagcacc caatagttgg aagatagtct 75420 gtcttcaaaa ttttaaacta catttatgtc caaaccagtg gcttttcctt ttaaaaaaat 75480 ttaaagataa tatgtgcaaa tcattttttt aaaattcaaa cagtatttaa gagtttcagt 75540 gaaacatgca ttttccttct accctggtac ttagttttac tccccaaggg caatcacttt 75600 ttactggttt ttagaaatat atccttcctg agatacttat gaatatccaa aagtgtgtgt 75660 gttgtgtata tcacctttta tatatcctgt ctctttacgt gcatgcattt taccgtataa 75720 actgttttct accctgcttt ttctatttga cctattttgg aaatgtcatt ttatttagaa 75780 cttcctcatt tattttaaca gctgcataat tagcagtaaa acttatgtaa gcagtccctt 75840 gtgaagggct gtgtcttttt gcgattatat ccggtgctat agtgtacatc cttgtgtgtg 75900 catcttggtg tgcctgtgct acgtatttct gtaggataaa tctgtaaaag tggaatcact 75960 aggtcagagg gtatggtcca ttttctttac ttatttattt tatttattta ttcatttatt 76020 tttgagacag agtcttgctc tgtcgcccag gctggagtgc agtggcatga tcttggctta 76080 ctgcaagctc cgcctcccgg gttcacacca ttctcctgcc tcagcctccg gagtagctgg 76140 gactacaggc gcccgccacc cacgcctggc taattttttt gtatttttag tagagacggg 76200 gtttcaccat gttagccagg atggtctcga tctcctgacc tcgtgatcca cccatctcag 76260 cctcccaaag tgctgggatt acaggtgtga gccaccgcgc ccggccccat tttattatct 76320 ttatttgctt ggatccttct tagcttcttc aatgttaaag atattgacag ttttcctctt 76380 actgaaattt ataaatccat tgactccctt gatattattg ccctggcctg actgattctt 76440 ctctctcctt tctcttctca ccccatgttg aggtccccaa ggtcacaccc agttttgatg 76500 actcaccagc atagagttgt acttgtgcct atgatttatt gcggtgaaag gatatagagc 76560 aaaattgcaa acggaaaggc acctggggtg aattccaggg gaaatccagt gcaagttcca 76620 aggtcgcctc ccagtggagt cacataggat gtgcttacat cctccagcaa ggagttgtga 76680 caacacttgt gaaatgtgga ctgccaggga agctcatcag agcctcagtg cctagggttt 76740 ttactggagg ctggtcacat aagcaccctc acacatatca aaaaattctg gtcccccaga 76800 aggaaagcag gtgtttagca taaccatatt atttgcatga acagttcagg tacaggaaat 76860 ccccgttacc agttaggttg gtgggtgccc ttctcaaatc ccaagttccc agacaccagc 76920 caggggcctg cctcgtaagg aggcctttcc aggacagcag tcaggcctgc caatgttaat 76980 tcttttctgc atacctccta attttagaaa ccaccgagcc tttgctgcct gacctgtcct 77040 gcttttcgat ttctttatct actttgatat ctttacaaat gatctttacc ctgactttta 77100 aatgtgtgct ctggccattc acctagcgtg tggttctgag tctccaagtc ttagcagatt 77160 tgctctcaga tgctctgcca acgcttcaca ccaagtatta caaactaaac tcgtcatctt 77220 cctcctgaaa cctgtctccc aggccaggcg cggtggctca cacttgtaat cccagtactt 77280 tgggaggccg aggtgggtgg atcacctgag gtcaggagtt cgagaccagc ctggccaact 77340 tggtaaaacc ccatctctac gaaaaataca aaaaaattag ccaggcgtgg tggcaggcac 77400 ctgtaatccc agctattcag gaggctgagg caggagaatc gcttgaaccc gggaggcgga 77460 gattgcagta agccgagatc acgccattgc actccagcct gggcaacaaa agtgaaactc 77520 catctcaaga aaaacaaaaa acaaaaaaca aaaaacctgt tttctcccca gctttgtcat 77580 gtatttagtg gccttatgta gacagtttcc tttgaaacat ctcttggact tctctgctct 77640 tccagggcca ttgccaccga cctggaatgt gtccttatcg tttcacgcca ggcttatggc 77700 agcagtcagt cacccagatg acctcctgac ctctggctta tttcaccccc actggactgt 77760 tgttcctaaa cacttctttc gtatgtcact ctaaaatctg accctggctg tacctttctt 77820 taactactcc ctgactgcgt gctgagagaa gatgggtctt gtcttttcct gcctctctgc 77880 ttttgtaaac tgccatttct acctgaagtg gcaactgaaa tcatatcttc ttcataaact 77940 gtctttggct acctcagtta gaattcctta tcccattttc ctgaagcatt tctttgactc 78000 ttctttactg ctcccccacc cttttttttt tctttgagac tgaattttgc ttgttgccca 78060 ggctggagtg caatggcccg atctcggctc attgcaacct ccgcctcctg ggttcaagtg 78120 attctcctgc ctcagcctcc tgagtagctg ggattacagt catgtgccac catgcccggc 78180 taattttgta tttttagtag agatggggtt tctccacgtt ggtcaggctg gtcttcaact 78240 cccaacctca agtgatctgc ccaccttcgc ctcccaaaat gctgggatta caggtgttag 78300 ccactgcgcc tgacccccat tttttttttt tttaaagatg ttgaattggt cagggtttgt 78360 agttacaagc aacagaagcc aactctttaa gcagaaaagg aatttgctaa atgatagtgc 78420 agagttctca gaatctctag caggatgaag aaccaggctt ggagaatagg tagccacaga 78480 tacacaagca tactgtagga cggttcccat gaagaggcat ctgttgtcac cactggacac 78540 agatggtact gtgtctctgc tactctacca atgccactgc tgtctctgac cccagatgta 78600 gctccctctg accctggatg cagctccctc tgaccctgga tgcagctccc tctgaccctc 78660 gatatagctg cccctgaccc cggatgtagc tgcctttgac cccagatgta gctttctcca 78720 aacccagata tagcggctgc ccccttgcca gagtgaatac tgcgtcattg tggcttcttc 78780 ttgtcactgg ttcttactta aaagctgagc tggaagttct aatgggcagt tttgtcacct 78840 gctcttacct tgttgcagtc tagatgaggt ctaatgttca taagctaggg gattttcaga 78900 tatggaaagg gataccaatt ttcagcagcc aaatagagta tcacattttc actccatgtt 78960 tcctgggtgt ctgttatgtt tcctgggtgt ctgactctta ggcttctttc aagctgcagt 79020 ctgcctaata gagagccttg catttaatca tcaaaaaggc aaagcaatat gaatcagcaa 79080 gggtgttttg gcaaataaca gcaaacctga ctgtggcgta agcttgtggt attgtctcca 79140 gtgtgatcag atctgtattt taatttttta aatgtaaatt aataatgatc tgtgaatcac 79200 caaagtagct tggagtagcc tagaaaacaa tgtatgtcct ccgttttcac agaagccaca 79260 tagtcgtggg ttaaatgagt cagcggcagg gcactgtgtc tcatagttaa aaaaaaaaaa 79320 aagtattact gaagtaatgc aggatctttt ctgaagtaga aggcatgatg aacccagaaa 79380 actaaagcag caagtggcca ccgttcttag catagttgtt tctcaaactg gaacaaccta 79440 taaacagttg tgaacaaggt attagaagtg atgggggccg ggtgcggtag cttctcccaa 79500 agctcattac ctcccaaagc aaccccagta ctttgggagg atcactttga gcccaggagt 79560 tccagaccag cccggccaac atggcaaaac cccatctctc taaaactaca aaaaattagc 79620 tgggcatggt ggcacatgcc tgtagtccca gctacttgga tggctgaggc aggagaatcg 79680 cttgaaccgg gaggcagagg ttgcagtgag ctgagatcac gccactgcac tctagcctgg 79740 gggacagagt gagtctctgt ctcaaaaaaa aaaaaaaaaa aaaaaagtga tgggaataga 79800 ttgttttgtc tcaaaaagct ctttccaaca ctaaaatgaa acatataatt aaaaatattt 79860 ttctggctat aaaaatatcg atgcttatta tagacatctg caaagtatga aaatatatga 79920 agaaaaaaat taaaatgcca tcatccccca tgaaaactat tgttatcatt tttgtctgat 79980 ttctttagtg tttctctttt tcttttttta atttttaatt tttttgagta tgtagtatgt 80040 atatctattt atggggtata tggcatattt tgatacagga tacagtgtgt attagcaagg 80100 ttttcttttt aatgtttata tttatttagt tgagatcata ctatatatgg ctctatagat 80160 tactttctct tatattacta acatttgtgt tattaaatat tctgcataaa gataatttta 80220 agatgaaatt tgatgttata aaaacttctc attttattaa gagattaacg ctatgaaacc 80280 tgctgctata tattcttgga accagctgtg acccaaaaga tcaatgtagg gatgtaggtc 80340 cttccccatt ctctacacac aaaatcagat actctgatgt gcagctgtag ccccagtcta 80400 cactgtctgt tgtatttttt gttttctggt gtcacgtgcc tcccaccctg ctcctagcaa 80460 ttgccatgac aacaaataga taattggctt ccgtaatttc tcatcttatt gcctaaggca 80520 acagagagct tgtgggctca gcttgcggtt cagcagctgc tttgttgcct ctcctctgta 80580 tgtgtgaggc ctgccagagc ccactttcca gacaggtgag agttcattca ttcaccatgc 80640 agttaccgat cgtctcttga cctgtgtcct ggggaggtaa aggtgacgag ccagttctgc 80700 cccatgcagc tcacagtcta ggcaaagcta catgcaaaca aacagaatcc aaagtgctat 80760 catgaaccct ctgagagggg ctgactcagc agcccaggga gcttgaagaa ggctccacag 80820 aggaggctgt gcctcaaggc gatttcggtt taggagccac caatttataa ccacttttct 80880 gtggcccgtc ttattttatt tcttatttct tgacaatcag aagtaccttg ggtaggtttt 80940 accatgcaca tcgtaatttg agtgagctta gtgtgaggct taacggtgtg tgggctgtac 81000 atcctggtca gatgctccag atggaggcag atggttgtga tgcaggagag gcagccacat 81060 agcacaggtc cccagccagt ggactgggaa gacagtgtag tcatctctgg ggaaggggaa 81120 tgacaagatc tggcagtgtg gcaggtccca gaaaaaaagg gctgggttct gggcagtgag 81180 ggtgcaggtt gagacctgaa tactgggtgg agccagctgt cagagtccac gcctgcagac 81240 tggactggtc cacggcaggt ggatgccatg tcttgaagac ccacaggcac ccactcatcc 81300 tcatgatcat gcagttctct ggtttctaac agtgcagtct gggttgcagt ctgggagtcc 81360 agcagagaag agcaggccct ggaatcccag gtgtgggggc gtggcttaac gtggagtttc 81420 cttcagaggc agtgagtgct tgtcattgtc tccgtcagca ttggctttgg gcctagtgtg 81480 gcctcgaacc ttctgttggg atcagcagtg gaacagtagg aaaaggaatg agtagacatg 81540 gcattgcaac aagtcttttt ttttttttct gttagaatta tcatattaag cagaagtttt 81600 gcttcacaaa ctctcagcca aatacaaaat actatgaata gtatttacct tgtgtctctt 81660 tccaaagaac tcatagtggt ttgcagctat tgcagatatc ctggccatgc ggtatgcggt 81720 tccttttttt tgtttttttt ttttttttga gacggagcct tgctctgtcg cccaggctgg 81780 agcatagtgg cgcgatctcg gctcactgca agctccgcct cccgggttcg tgccattctc 81840 ctgcctcagc ctcccgagta gctgggacta caggcgcccg ccaccacacc cggctaattt 81900 ttgtattttt agtagagacg gggtttcacc gtgttagcca ggatggtctc gatctcctga 81960 cctcgtgatc ctcctgcctc ggcctcccaa agtgctggga ttacaggcgt gagccaccgc 82020 gcccggccgc agttcctttt tatagctgtt tgaataggaa agatgacttg gaaaatgctg 82080 gattctgaga tttatgtgca gccttaaaaa gtgtagtttt tctctatcaa taatgagtgt 82140 gggttgtaat tgcttagtaa gtaattttgt ttatgtaaac gtacatttgt taaatttttt 82200 ttcttaggta atcccgtatg ttggcaccat tccygatcag ctggatcctg gaactttgat 82260 tgtgatatgt gggcatgttc ctagtgacgc agacaggtaa aatcactgtg ctaaaggaag 82320 gagcatgaat aggctgtctt tttgtgattg tggaatgata acagagtaag gcgggagaga 82380 ccatttgata ctytgaggcc caattagctt tcatcagcag ccctggccaa ggtgctgagg 82440 agattggaat gaatgactaa ataaaggtta ttgggattta tttcattgct gtaagtctga 82500 tttcagtata aaaaaattag aactatcagc tggatgtggt gacttacaca tacttttcca 82560 gcactttggg aggccaaggc gggaggattg tttgaggcca ggagttcgag accagcctgg 82620 gcaacatagt gagacccccc ccatctgtta aaaaaaaaaa aaaaattaaa aattaactgg 82680 gcttggtggt gtgcgcctgt agttgtagct actcaggggg ctgaggtagg aggatccctt 82740 gagcccagga gtttgaggtt gcagtgagct gtgatggagc cactgcacta tagcctgggt 82800 gacaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaagaaaga aaaagaatta ggtatgtcat 82860 taaagaaagg aattgtggtc agatgacagg gagagtctag ttttagtctg acattcccac 82920 agcatcacag atctagttca gatggtttta ctgaatactt gctttggata caagctgtgg 82980 tatcattagt gttgggctca gctctgtgta cctaacacct gaagagcagt ggtttaagat 83040 gtgaaaatta agtctcaagg agacagccca ggccttttca gttaactcct tcaagtcgtt 83100 agagaagtag actccttcca gcttaccact ctgctatctt gagggtgagg tgaggtcccc 83160 tttcccatta tccttggcag ctagatttcc agccctcact tctgtgcttt gggtagctgg 83220 atgggtgcat gtggtgtttg cggggaaaca gagctggaca aaaggcaagt gcttgctgac 83280 ttttaaggca gtttctagta gccttccctg agcacttcac ttccatctta tcagcagagc 83340 tttagctgca caggcaggcc tagctgcgag ggaggctggg aaaggtaggt ttttattctg 83400 ggcagattca gacccagttc aaactcaggg gctattttac tgaggaagac agaaaagatt 83460 agacagtcag ctctttaggc ctcatagtga atgaatgagg agggattggt cagtcccttg 83520 tcactgggcc tggagtgtag tgcctgctgg gcctttactg gtggctttcc tttctgagca 83580 ctcatgaggc ccctgtgtct tccctcatat agattccagg tggatctgca gaatggcagc 83640 agtgtgaaac ctcgagccga tgtggccttt catttcaatc ctcgtttcaa aagggccggc 83700 tgcattgttt gcaatacttt gataaatgaa aaatggggac gggaagagat cacctatgac 83760 acgcctttca aaagagaaaa gtcttttgag atcgtgatta tggtgctaaa ggacaaattc 83820 caggtaggtt ttggagaggg acaggttgag tcctcattag tgagcaggag tgcacagggg 83880 ggcctttcac atttgtgagc ccagccttgt atttcctaca cctgagatat agtttggctt 83940 tgtagtcttt ctccataaaa ggaccaggaa ggcacctaaa tatgaggggg tggcaccact 84000 actctccagc cagttgttgc catgcagaaa tatggtccac tgtgactaga tctttttatt 84060 agatcctatt tctcctagca gggctgagtt ctgaattgac acagtattat gttcatgatg 84120 ggagggtaag ttataatata accgtcacca cctgaagaac taacaagggc aatcccagca 84180 tagaaatcag aagggttttg taaattcaag tcttgccaca agacagttct gtaggatcat 84240 gagattttta gacccagagg acatcctaga aatccttgat gtcagttcca tctctggctt 84300 catggagtgt cttataccta gcgcgcgtgt gtatggttga atttggtccc agaagctctt 84360 acacctgctg gccctctggc ctgtggagct ttcccacagt agaggtttgt accaacgtga 84420 gagaagactc acatgcctct ggcacagatc ctttctgatc ttcgggatac tgctcctgcc 84480 cgaaagtctt tctgaatctc ccaaactcca ttcacctctc ccttctctgg ccttttgagc 84540 ccgtgtctgt atcattcttt ttcacagttt ttaacagttg tgctttggct ttatgtgttt 84600 attttgcctc cacaatggga tttaaagctc cttgagtcag agactatatt gtatgctgct 84660 cgcgttttct gcctataacc taacgtggta cctggcattt gagagggagg gagggaggag 84720 gctcgtagcg tgccgaggac ctgcagaagc tactttctcg tcatcttact gtagtctgtt 84780 gaggtagaga ttgttcctac ttcagaataa gaaaaccgaa ttcaaatatg ttgggtaact 84840 tgtccatatt aatttattta gcaaatacaa cagattttga gtgtctgcca catgggtggt 84900 ctccagggac agtgttgtgg ggagctcgca ggcagatctt taacctgggt tcacaatctc 84960 cagggcacct gtgcctgggc ttccaggcga ccttcgaacc cagatgtctc acatgtatgc 85020 agaggcgcac acaagcacac gcacatatac ttatgactgc ctgtttgtct ggggagagac 85080 agttcctggt gcttaatcaa atcaggaact caaaagaagt tcggaagcac tgctggtgtt 85140 ttgggtgctt tcggttacca tttggtcacg tgtgtggaga cctgtgggaa caggtataaa 85200 attggacgca aggaaacatt taaatttgga taataagtta atttattaac tgtttttttt 85260 tggtggcggg ggggctctgt cttctgtatc tctctaggtg gctgtaaatg gaaaacatac 85320 tctgctctat ggccacagga tcggcccaga gaaaatagac actctgggca tttatggcaa 85380 agtgaatatt cactcaattg gttttagctt cagctcggtg agtgaccttc cacagcttgg 85440 ggtcttttat gaggatggtt tctgatgaga tggtagaaaa aatcttcaaa taacacttct 85500 attgacataa aaaggacgta tctccctgac tgtagtatta attttttgga agtgaactgt 85560 tcacactagc agaaggctgt ttatcagcca gggcttcatt gtctgtagga tctcaaacct 85620 agtgtggttt taataaaaca cacacagttt ttagctgggt agcagctatt tcctttgcat 85680 gggcataaaa tggagtattt ctgtaagaca ggttcctagg ctgggagtgt ctgagtcaaa 85740 gagcacagtc atgtgttgca taaggacagt tcagtcaaag atgaaccgca tatacaaccg 85800 tggtcccata agattgtcat atactgtatt tttaccatac cttttctatg tttaggtaag 85860 tttatatgca caaatactta ccatcctgct ctggttgcct acagtatttg gtacagtgcc 85920 tgctgtacag attcactggc caggagctat aggccacacc ctacagccta ggtgtgtagt 85980 tggcagtacc atctaaggtt gttaagtaat attctgtgat gtttgcacga tgacaaaagt 86040 catgtaagga cacatttctc agaacatacc cccttcgtta agcaacacat gactgtcttt 86100 gcattgaaaa ttttgataga tactaactcg cccttcacaa gggtaaaaac agtttgcact 86160 ctcaacagcc atgctcccac cttcttgttg acattacatc ttattctctg taatgtttgc 86220 caatctgatg gggggcggaa aggaccccag tgtcaagtat atttttggat catagttttc 86280 aagcatattt ttagtaccat ttataatttt tttatatgtc aaacaggtta tatatagaaa 86340 atattttcta ctggatgtta caaattaatc tttattatct tttctcagga cttacaaagt 86400 acccaagcat ctagtctgga actgacagag ataagtagag aaaatgtaaa tattaaatct 86460 tttaatgagc cactggttta aaaatgttgt tttagctgcc atgttaatga aatggcaaga 86520 aggctgggtt tttgaaaatt atgcttttag aacgcaagta atcacttgaa aattgagata 86580 catacttgtg gtgccaggca cgcagtaagt ttttgctgat gattcacctg tcagtttctg 86640 taactgccac tcactgttct tatgtaaaaa gcactctctc actcttaact gctgaatagt 86700 actgttctgg ggtatttcca aatattgaac atcagccagt gcactggcaa atgaacttcc 86760 atgtgtatct tcaacccctg ggagaataac tgcaatttaa aaatgcgctg ttattaatgg 86820 agaaagtgag gtcttaccga ctggcacgtt cacacctcac agacagaata gaatcttagc 86880 attctggggg caccctggaa aggacaacta agacacgttt gaagttcatg tagtgctggg 86940 tgaaggtggt ggctcaggcc tgtagtccca gcgctttggc tgaggtgggg ggattgcttg 87000 agcctaggag tttgagatca gcctgggcca catagggaga accccatctc tacaaaaaat 87060 taaaaaatta tctgggcatg gtggcgcatg gctgtgatcc cagctttggg tggctgaagt 87120 aggcggatga cttgagccca ggaggttgag gctgcagtga gccatgattg agccactgca 87180 tcccagtgtg gatgacagag taagaccctg tctcttaaaa aaatttcata tagttctatg 87240 aaaaattatt aatttatggt ggaggataaa ggactcagat gaacagggat atcagactct 87300 cttctcaacc cgtgtagccc ttcacaacac cataccattc cgtcataaag caccagctgc 87360 ctggaggtca caccagagtg gagcaggaac atcccaggct ccggccaggc tcagctcagc 87420 acaaccaaga cttcagatta taaactataa ttcttcccct tctaacattg ttgtgttttg 87480 tttcttttcc aataggttcc aaagtctggc acgccccagc ttgtgagtat ttttgcctgg 87540 gttatttcat gtggaatatt ttataaagtt gcatagaaaa tgaacagttt aaaccgtgga 87600 gggcagcttc attcattcca ttccttactg tagaactgtt tccctacagc ctagtaatag 87660 aggaggagac atttctaaaa tcgcacccag aactgtctac accaagagca aagattcgac 87720 tgtcaatcac actttgactt gcaccaaaat accacctatg aactatgtgt caaaggtttg 87780 aagagcccca aattttctta actctgtata aaaattaagt tgtaatgagc tgttacgagt 87840 aacctgtatc cacaatagaa gcccaaagca gccccctctg catttgtgtg ccgtccctgg 87900 atggattcga gagtcaacca ggcctgcctc tgagccattc ctgtgtattt cctcagcacc 87960 tccctgcttg gctgcttccc cttcaggcag aacacagtac tgcctcagac cccaggcaca 88020 gggggccttc ctggcgtgtt tcactcatac agagggcatc gggtcccacc ctgtcactca 88080 tttcatcgtc taaaatgtaa tcatgagtgt ttgcttcgag ccagggacag tgctgctgca 88140 ggggacccag ctgggaccaa ggcagactgt ctctcccctc ctgggattta cagggtcatg 88200 gctctgaaac attctgtagt gttctttgga cacgagtttt ccctggagat cgctttctgc 88260 aggcctcttg gtcctgactg tggcttcttt tcagagcctg ccattcgctg caaggttgaa 88320 cacccccatg ggccctggac gaactgtcgt cgttaaagga gaagtgaatg caaatgccaa 88380 aaggtcagta tccttcggta ccagtcacag tgcagatact tccgtgcctg ttaccgcctt 88440 ctacccgtga acggtcctgt gagctggaag tagggctagt gtcagaatct tcatttccaa 88500 agtgagatga ttcaagcagg aggtggttag attgtgaaca gccagtgggc agcagagccg 88560 actaaggccg tgttctgacc tcggcttttt ctggccagac aagagagtag catttttgtc 88620 cacgaggcct atccttgcct tgtagaactc cagagcagcc ccgtaagatc aggcaacatc 88680 ttttcttttt ttttttgaga tggagcctca ctgtgtcacc caggctggag tgcagtgtca 88740 caatcccagc acaccacaac ctccgcttcc tgggtgttca agccattctc ctacctcagc 88800 ctctggagta gctgggatta caggcgcacc accacgccca gctacttttt gaatttttgt 88860 atttttagta gagacagggg ttcaccatgt tggccaggct ggtctggaac tcctgacctc 88920 aagtgatccg cccacttcgc cctcccagtg ctgggattac aggtgtgagt cgccacgccc 88980 agcccaggca acatttttta gggcccctct tgtcatgtga tttagaaaat ttctgcttta 89040 acaacttttt ccacagacgt ccagccttct gaaagcttga aattagagct atttcctaga 89100 aagtggcata ctttcaagaa ggaaggaaca cgggtagatg atgaaaagag aatacctgct 89160 tgagaggatc ccaggctcct gcagcctgaa gtagtcattc agtttagcgt taaaccttcc 89220 atttctgtcc aaccacatct cagcctcaat gctgatttta aaggggtttt ttttttcgta 89280 tttttatttt gcaagtaacg aattagtgga atgctgactg ggtttaaaat ttcaacttca 89340 cctgcattcc catgtccatg tggatacgtg tgtttcatag agttagaatc atagttcaag 89400 tctggtcact aacattgctg aaattgccac tactctgtcc tacttggtta attaaggttt 89460 tttttttctt tctttctcaa aagctttaat gttgacctac tagcaggaaa atcaaaggat 89520 attgctctac acttgaaccc acgcctgaat attaaagcat ttgtaagaaa ttcttttctt 89580 caggagtcct ggggagaaga agagagaaat attacctctt tcccatttag tcctgggatg 89640 tactttgagg tgaggttcca gtttttgaaa atgggacagc aataagaatc ctgggagcag 89700 gggtgggata agtggtccat ttaaatcaag tcctaactca gtatgtggag gttgtgtatg 89760 ttttttgttt acttggagat tgtaatttgc cccttccttt ttataacgtg ggcaatcagt 89820 ataaatggca aagccagtag agtgtcaaat tatgcacatt ggaattgaca tttgtcatca 89880 tattaaaatt cctgtgtagc cccatattga taggaattta accaggaagc ttgtctcagg 89940 actggagtca cacatttaat catataagca gacttgagga ctggagaccc taaaactgct 90000 tgcttgcact ggccatcatc tcccatcagg gtaggtggca gtcctttctc ctaaggagtt 90060 agtcttgttt atatgtattc aaggaaaaat acatcagtcc cttggaacta aaaggcatgc 90120 agtcctgagt ccccagatag gtgaatattg taacacatac ctttcccgaa atatgtttct 90180 gggatgctga gcagagaata gtctccttgt gatgtggatg ccgggtgttt ggccagcctc 90240 aatcaccagc tcaggtgcca ctgcctcaca cagtcactta gggtcattgg tttaggttat 90300 cattctacag cattttaaac tgacacattg tctggaccat gtgggttctt gaggactcat 90360 caaaacccgt tactaaaagc atgaatatca ggcgaaatag atagcaatgt gacattcgta 90420 tttatcccta agttccagtc taatgcagtg ccctggtatg tggagtgtag acagatgtgg 90480 gctaatcatg gaaggttccc tggaagttgt ggatattggt ttcgaattca gaaagctggg 90540 aaggatgtgg aaggctgaag gttggctttt ctagatttag ggcatgattt gaacaagtcc 90600 ttagaggtgg gaagggcagc acagggttgt tggcttggca agagtcaagg tgcaaagggt 90660 gacttggggt tcactggagg gaaacagaga tgagtgctct agaaggaagt tgagccttgt 90720 ggtgggtgac aggaaaccaa tgatgtaact tgtttttgac ctatctgggc cccaagtttg 90780 gatctgctat attaatataa aaaaggataa taatgataca ttcaaataat gctgaaaaat 90840 actaagatga aaatacctcc aacttcgtaa ttcaaaccat accattagga ttaggtgaac 90900 cacattccag gcgttttttt gcagagacag tgaaagggat ggctggctga aggaatgaat 90960 agatgaatgt tatatgcttt tgaacaatcg tcttttccat ttaattttct aattcaggag 91020 cagtaattat ccttgtgttg atcactgctg acgattttct atactgatag gtcctttccg 91080 ggggcttcca tctcttgcct tttaaatatg cttgcattga gattatctca ggtctttcca 91140 ttatgccatt actttcattt taaatcttct tgctctttca aatacacttt agttgtatct 91200 acagtgtttt aaaaacaatc tcattcagtg ttgtaatttc atctgtgggc tcttcctctg 91260 gatgaaatcc gtgttcctcc cagctgttcg gcagcatcag atggttgtga gggattctgt 91320 tgttctgttt tcttctaggc aaaggatgtg ccttcttttc atttgcagta gtctgctcac 91380 ccggaagcat gtcatttctt tgccacttgc ttgtaattca ctggctttgc acttgctctg 91440 atacagtaca ggtaactaat tgactccctc tgctgccaac ttggttttcc ttctgagcta 91500 tagcatcagg ctgtgtgttt tgtgttttct tgagattttg ttaaatatat ctggggtccc 91560 ttctacctgg ttggaactgg gattcccacc attcttgtgg ggatagaatc tcaggttaca 91620 cctatttccc caatcctctg tagccacaga agcttcatct tggccagctc tgttatcaga 91680 gtgcaggact tgggctgaaa tttcctcccc ttcctgattt tccttgacag tcctttccac 91740 tgctcctatc aatcaaaaga atgaaaaccc tcaacttgct gctttgcaga ttcaggtttt 91800 gtgcttcttt ctggcctctc ggggtggggc cgggttagca gcaaggctga gctgcccctc 91860 tttcttctga agccttcatg ggggcgagga gcacagggag agctcagtgc agggcctccc 91920 agtggccttc tcagagtggg tggaaaccca gcctggcact ggcagcgtgg caccagaagt 91980 atgaagtgta ggtgtaaagg tgatgtaaaa ggctagtagg ttttttggtt tttcattgtt 92040 tgagttttgg gcatagatga ctgtgaaggg cgaacactgc cgatggatct gaatgaattt 92100 gtagtatgtg caccacttcc aacttacggg atacccagct ttgacggctt tggacaaaca 92160 cactgaggcc aagatgtgct gagcttatca ggatcaggat caccaagcag ctgtaaaaac 92220 cctagcaagt gccttaagct gctgaaattt catattaatt gtctggtttg ttcatggtcc 92280 tagagtttga ggcagaaaag tcaggatcca agtcccttgg ttccaggcta cagctggaaa 92340 cagcatctcg gtgaactaaa gcaaccatat taggagtttt cctgctttag gagagtcccc 92400 agcatcggcg aggagggggc agcactctgg ctttccagga gcaaggggca ggatgcggcc 92460 gagggagagg ggctgtgttg aggaaaggag ggccgcaggc cctggggatg gtgtgaggct 92520 ccaaacatgt ccgagtcact tccctgggtg ggatgaggca gacagtgcca ccaccaggga 92580 cactttagtt agattagggt cttggaagtc acagaaggaa gtcagcagca gcaggctgga 92640 acttttctat gtataatcaa atggtttact ctgacaccgt tagcatgtaa caaacacaaa 92700 attttaaact aaggggaacc actaatggca tgtttccttt cctttcagat gataatttac 92760 tgtgatgtta gagaattcaa ggttgcagta aatggcgtac acagcctgga gtacaaacac 92820 agatttaaag agctcagcag tattgacacg ctggaaatta atggagacat ccacttactg 92880 gaagtaagga gctggtagcc tacctacaca gctgctacaa aaaccaaaat acagaatggc 92940 ttctgtgata ctggccttgc tgaaacgcat ctcactgtca ttctattgtt tatattgtta 93000 aaatgagctt gtgcaccatt agatcctgct gggtgttctc agtccttgcc atgaagtatg 93060 gtggtgtcta gcactgaatg gggaaactgg gggcagcaac acttatagcc agttaaagcc 93120 actctgccct ctctcctact ttggctgact cttcaagaat gccattcaac aagtatttat 93180 ggagtaccta ctataataca gtagctaaca tgtattgagc acagattttt tttggtaaaa 93240 ctgtgaggag ctaggatata tacttggtga aacaaaccag tatgttccct gttctcttga 93300 gcttcgactc ttctgtgctc tattgctgcg cactgctttt tctacaggca ttacatcaac 93360 tcctaagggg tcctctggga ttagttaagc agctattaaa tcacccgaag acactaattt 93420 acagaagaca caactccttc cccagtgatc actgtcataa ccagtgctct accgtatccc 93480 atcactgagg actgatgttg actgacatca ttttatcgta ataaacatgt ggctctatta 93540 gctgcaagct ttaccaagta attggcatga catctgagca cagaaattaa ggcaaaaaac 93600 caaagcaaaa caaatacatg gtgctgaaat taacttgatg ccaagcccaa ggcagctgat 93660 ttctgtgtat ttgaacttag ggcaaatcag agtctacaca gacgcctaca gaaagtttca 93720 ggaagaggca agatgcattc aatttgaaag atatttatgg gcaacaaagt aaggtcagga 93780 ttagacttca ggcattcata aggcaggcac tatcagaaag tgtacgccaa ctaagggacc 93840 cacaaagcag gcagaggtaa tgcagaaatc tgttttgttc ccatgaaatc accaatcaag 93900 gcctccgttc ttctaaagat tagtccatca tcattagcaa ctgagatcaa agcactcttc 93960 cactttacgt gattaaaatc aaacctgtat cagcaagtta aatggttcca tttctgtgat 94020 ttttctatta tttgagggga gttggcagaa gttccatgta tatgggatct ttacaggtca 94080 gatcttgtta caggaaattt caaaggtttg ggagtgggga gggaaaaaag ctcagtcagt 94140 gaggatcatt ttatcacatt agactggggc agaactctgc caggatttag gaatattttc 94200 agaacagatt ttagatatta tttctatcca tatattgaaa agaataccat tgtcaatctt 94260 atttttttaa aagtactcag tgtagaaatt gctagccctt aattcttttc cagcttttca 94320 tattaatgta tgcagagtct caccaagctc aaagacactg gttgggggtg gagggtgcca 94380 cagggaaagc tgtagaaggc aagaagactc gagaatcccc cagagttatt tttctccata 94440 aagaccatca gagtgcttaa ctgagctgtt ggagactgtg aggcatttag gaaaaaaata 94500 gcccactcac atcattcctt gtaagtctta agttcatttt cattttacgt ggaggaaaaa 94560 aatttaaaaa gctattagta tttattaatg aattttactg agacatttct tagaaatatg 94620 cacttctata ctagcaagct ctgtctctaa aatgcaagtt ggccttttgc ttgccacatt 94680 tctgcattaa acttctatat tagcttcaaa ggcttttaaa ctcaatgcga acattctacg 94740 ggatgttctt agatgccttt aaaaaggggg cagatctaat tttatttgaa ccctcacttt 94800 ccaacttcac catgacccag tactagagat tagggcactt caaagcattg aaaaaaatct 94860 actgatactt actttcttag acaagtagtt cttagttaac caccaatgga actgggttca 94920 ttctgaatcc tggaggagct tcctcgtgcc acccagtgtt tctgggccct ctgtgtgagc 94980 agccaggtat gagctgtttt agaagcagcg tgttgccttc atctctcccg tttcccaaaa 95040 gaacaaagga taaaggtgac agtcacactc ctgggttaaa aaaagcattc cagaaccact 95100 tctctttatg ggcacaacaa agaaacgaag gctgaagttc gcctacccaa aatgaaaagt 95160 aggctttaca gtcaaaagta cttctgttga ttgctaaata acttcatttt cttgaaatag 95220 agcaactttg agtgaaatct gcaacatgga taccatgtat ataagatact gctgtacaga 95280 agagttaagg cttacagtgc aaatgaggcg tcagctttgg gtgctaaaat taacaagtct 95340 aatattatta ccatcaatca ggaagagaat aataaatgtt taaacaaaca cagcagtctg 95400 tataaaaata ccgtgtatca tttactcttt ctgcagctct atacgatagg caggagaggc 95460 ttatgtggca gcacaagcca ggtggggatt ttgtaacgaa gtgataaaac atttgtaagt 95520 aatccaagta ggtgtattaa ggcaccaaaa gtaacatggc acccaacacc caaaaataaa 95580 aatatgaaat atgagtgtga actctgagta gagtatgaaa caccacagaa agtcttagaa 95640 atagctctgg agtggctctc ccaggacagt ttccagttgc tgaatagtct tttggcactg 95700 atgttctact tcttcacatt catctaaaaa aaaaaaaaaa aaaaatcaaa attaaaatct 95760 gagtcagtct gcctgcctcg gttctcatta gtttaattct taatgccttg cactttccag 95820 caatcattca atcaaaagag tgaaatgaag cacattaaca aagcaggagg cgccacggac 95880 cgcctccctc cacaccgctc cttccgcctt cattccttgc ccacaggctt gcactggaag 95940 ctgaataaga atccccaaaa ctcaaacttc ctagggatgc caccccttta gtagctcaca 96000 cctcccccct ccaagagcta agaaacaaag gagaatgtac ttttgtagct tagataagca 96060 atgaatcagt aaaggactga tctacttgct ccaccacccc tcccttaata ataacattta 96120 ctgttatttc ctgggcctaa gacttatgtt ccagaactgt cacagctccc catgtcacac 96180 ccactagctt gtgatctttg tcaaataact gaaatctttt aagcctctag tttcttcctt 96240 tgtaaaacag agataaaatg ttgtggtttt taagtgagat aatccaagta aagcacctaa 96300 catggagtag tgaatgaaca tcggttgcta ctaaaagtgg acatcctacc gcatccttaa 96360 tgccactagg catttccata caatctgggg accaaaactt caatcatata aatgtatgag 96420 gttaattaaa aacactactg taatctgctt gtatgatcac aaaccaccac aaaagaaaag 96480 atcgtgaaga ttacactgta aacggactct caaatgatca ggaggtggtc acttcgcaac 96540 ttgctccctc cacccaactc aaaacaggag ctcgagcctg cctgtatttg agactggagc 96600 tgcctgtatg aggactggat caactgctag tcacgttata tccaaatctg cattatcatt 96660 gggcacattt tcacagaatt ttactgaatt attccttaat tgtttaatgg ttgggaatag 96720 tttgggaatt accttccatc aactctgcta agaaaggaat ggattctggt agcaagacaa 96780 tataattctc ctttagtttt tcagccagtg ctaacacagt aatcaaagca gcaaatcgaa 96840 cctgaaaggg ataaaagagc aaagaaataa aaagtagtgt tactgtattt attatcttaa 96900 gagctgtact gacttgagac aagctctaac tttttaaaca ttagttcaca cgcgtttatt 96960 cacttcatta tgttcattaa gctttcatct tagaatacca gtttcaccat ttgggagctg 97020 tttgtaatat gtgcaacctt ataaatagtg ttttccaaac tgtgtcccag gactgcaaat 97080 ctttaatgtg aaatgtcttt ttataatctc ttcctttaaa aaaaaccaat aaaataaaat 97140 gccacatgca aactcaagtg tgtcaccaga ttttacttca ttggcgctcg ccagcccgcc 97200 aggctggcaa taaagtgcct ccagccacct ctggcaggtc tcctcaccca cagcccctga 97260 ctggtcacca ctatagttgt atgaggggcc aggacaatcg cttgggataa actcccatct 97320 cagcactgaa taaaaaacat tctgtgtcac aatatcctag ttttggggct ttaaaaacgt 97380 ctaggtgttc ctcacatgcc ttgtctataa taaggaaagc aagcagtagt tgggtattgt 97440 tagcttttga aacaaaagcc ctactggtct tctaattttg gatattttaa ttaaagaata 97500 tctggacagt acaaagtgaa ttattaaaaa accatttgta actacctaga ttcaatcagg 97560 atttccttga tttgtgcaaa gtaaaatatt acaataaatt tgatactgct acttgtataa 97620 aaacctatgg tttaaaatgt gggggttcat cataatagtc tcattgttag catatcctaa 97680 taaagaattt gaactaataa atcctattaa taaaattctg ctttggtctg ttatagccag 97740 taaagttcta atacaatcat tagtttgaga aatggtgact cattgctaaa acagtttgaa 97800 atttgtaaca cttgggtgtc aaattttgac ttccactcaa cctacccatg ttttatttcc 97860 actgccacca cttactcaac aagatcataa gcctagtatc tataaacaac agaatgtatt 97920 gctctaactc aaaagactat agtgtggata aattcaatgc atttctctct ggagcacaat 97980 gacatttcaa tagcacttaa aaaagaagga attacttcaa atctttgtta tttaaaagta 98040 tttagaaagt attttagtac ttctgcccaa cgcaccattg gggtggggat agggcattgc 98100 tattctttac aaatagccta taagtaaaaa acaaaatttt cttaggcaca aatttctgcc 98160 taatacaaaa gaccagacct ctagtactgg atgacaaata gcaatgttct tccctgccag 98220 tttactaggg ggcctacatc tgtgaccacc tgcaggctgt ttaggctatg cagtgaaaag 98280 atgcagtttc agtacttgtc acgcagttcc taaccttagg cgaggagtct ctcgtcttta 98340 gcagaatctg gtagttcagt ggtttccaaa gagagtcatc cgccatggcc actgaaaact 98400 gtgcgatgca tggtatcagg tgctttgtca cccgttcctg gaatttctct tctcccccaa 98460 gcctgttttc cagctaggaa gagtaagaca aagactttga acaacaagtc tcatttcttt 98520 cttctgtttg aaaaaatgtc caacatacaa atattttact atctttcatg atattagcag 98580 gttcaaaaac caggcattat tctaatactc tctagggcaa atgtattgcc ttctagaact 98640 caaatggaat ctcataccct ttatcatcgc ccctttctct ccagcagaac atctcagagg 98700 agctctttgc tccagaggac agccatgctc tgacacgttc tcagtgaggc ccagttaaaa 98760 caaatgaata cattaaccat gacagcttat atcatgtctg tcttttgagc agtttaaaaa 98820 ataaaaaata aaaaataact cagggccagg catggtggct cacgcctgta atcccagcag 98880 tttgggaggc caaggtgggt ggatcacttg aggtcaggag ttcgagacca gcctggccaa 98940 catggcaaaa cctcatccct actaaaaata caaaaattag ccaggtgtgg aggcgggcgc 99000 ctgtgatccc agctattcgg gaggctgagg cacaagaatt gcttgaaccc gggaggtgga 99060 ggttgcagcg agccgagatt gcaccactgc actccagcct gggtgacaga gcaagaccct 99120 gtctcaaaac aacaaaacaa aactcaaatt ccacaatgaa gttatatctt tgaaaaaaca 99180 attttcaaat aaaacatttc attaaaaaga ccagaaaaaa caaccttaca aagaaaaatc 99240 ctagcaagct gtcatttgag cagatctaaa acctgccaag ctcgaacagt gatggcttcc 99300 tcagcaacga aagatgattc tgtttggtta cctgatccac cagaggcatc atcaaggctc 99360 ctgctctctc tttacttata aaatgctggg tatcaaaaag gaagattttg tataaacagt 99420 tcaaaataaa ctgcaacagc aagcagcact tttcagggtc attttcagag tcaaaaaatg 99480 cttcatctgt agacgtggga agagtaaaaa tgaaaaaaca ctgaacttaa ccatttaatc 99540 tccaatgttt acattgaaat cactattaaa ataactaaat cagaagagtc taaaatgatc 99600 tagaaatcat aatcaggacg aaggcagaac acaatggatg gtctctcgaa gaatgattcc 99660 ttcttttaga gttaagattc taacactcac tctggcaagt taaattccct caactgtcaa 99720 gtgggtcacg tattagcatt agagaataaa ctaatcttaa tttttgcgtt ttaaagttac 99780 ttccagtaac tgacagtaac ggccatttac tttattcttt ctcccaagtg aggtgactta 99840 taacattcgc tcatcatgct aaaacaacac ttcactgtct gacaacaatg aagtaaaaaa 99900 ttcaccctcc ttagcttagg acttaagaac ctctaaaatc ttgcttccaa gcactagctt 99960 gtgtcttact ggtaccttgt ataaggcaca caggacaagg gtgacagctg aactgaagcg 100020 accacccacc tgttttggag atgttcacct ggtccaaggt gtcagcaaaa ggcttcacta 100080 agtggccggc aaacagagta aaaagccctt tcagcttttc agcaatgcaa tctgccaagt 100140 tgtaaaatgt caacaacctg tcctttgggg catcttctgt tttagcccaa tcaaacagct 100200 gaaaggataa gacagtatta gtttcttcga catcttgtca cttaaatctg agcacaaaag 100260 agaggaagag gaagaaagcg tcaccttgaa gaacaggggc ctgaatgtga cctcggaaag 100320 tttgacaacc atggctacta gacagtcaat gatacaattt tccgtttttc caacttcctc 100380 cagatcgttc tgaaaacaga agagcccatt tattagagtg ctgatacctg actgtaaatt 100440 attttggcaa gtaccactgt tacacggcta gattgttctc ggactcttca ataggtggat 100500 aacagcttta ggatttggag gagtgaacct gagcttacct cagagtgctg ggctcggaag 100560 tccagggcct ccaggaaaaa ggcggttagc tgagactgat gggaggtgag ctcttccttc 100620 ttcatcgccc caatatgctc ttgcaagatg ctcataaacg gacccatgtg attctaccaa 100680 taacacagga aaaagatgtg ccattttcaa atgattccta gagttcagcg gtgtgtattt 100740 ttaaaaacta aatcttcttc tttaagtcaa agtttacaca ttgcagtacc acctctccct 100800 tctccaaagt cttaataccc aataagatct aaccttccag ttcttctcaa tctgcttgta 100860 agtttttttg atggcgggca acaggactcg gggtgcaagt gtggtagcca gtgtcttttt 100920 aagagatgtg agacggatat tagcctgtga cgcagaaccc atttcactag tgattttctc 100980 cagatgaatc acctacagga atataaaaaa agtgatcagg gccactgcag atcttcgctg 101040 acaaacacac acttacagag aggcttcatg atgaggtact agtgtttgga aaatgcttag 101100 cactttttaa ctacacacag agttcctttt aaagtcagcc ctaaacgtca gtggataaaa 101160 ctgggcagac acctcttgcc caacttgcga tcagggacga aggccgatgg tagacgcaga 101220 cgcacacaca gcacccagac agatgatttt cttagaggac aggaatgcaa gggaccacgg 101280 caagagtcaa gttgctaaaa aactgagaaa gctcctcaga gcacaggccc ctttctctga 101340 gaaggctact tttaaaccct ggctgtggtg taagtgaagc ggtttaatca tttgccccat 101400 ggtaatgaag gctcctaacc ttgtaaatgg caaatgatca acacaatgga acagccaggt 101460 ctcaacactc ttgagcatct tcaatcataa ataccactgg cccctagcgt gttgacagga 101520 aaccgctgac gtgcaataca aaaattctgc tttgcaagat gccttaggat taaacctctc 101580 acagtagaaa cagggcccat caatttccac aagtaataaa aggcggctct accagcccaa 101640 ctccaaagat ctcacagaag aaaaaaaagc cagaatacat tccgcacaat taaagaagag 101700 aagcatctcg ctaaaaagtg acccccatat caatttcaag attaagtggc aaggatgatg 101760 gaagagaaaa agtacacatt taataaaagc aagcacatct cttcagaaat aagactcctt 101820 tctgtcaaac ggaaactaac ccttaaagaa aaaacaaaat cactacattt gtgatctttt 101880 accttcccca gccaccctgc gtagcatgtc gtggctatcg tggctcacct gggagagaat 101940 gccttccaga taggggctga tgaagtgcgg gagagtctcc acaaccttct gcagagcagc 102000 caaggcactg agcaggtaga cctcgctgga gaccagctcg ctggtgttct tcattgttgt 102060 cagcaacgat ggcatcaggc tagaaacaaa gtaagagctt tagaagaact tgaagcagaa 102120 acagaggcta gggaatggag tagagggcat tatgaaaaaa accagcaaac tgtgcctatt 102180 acatcgctat ctgcctcata gcctaaaaag cagtgtctat acattttatg tggctaagca 102240 caagaaatct cccagtgcta acagtatgga cacaacagta atttaaaaaa taacaatgtc 102300 tttcattaac tgaacactta ctatgtgtca ggcactatgc aaaactcctt gcaagcactg 102360 ccctacagaa atcctatgag gtagatactg tctctgtttt atagacagca aagctctaac 102420 aggttaagga acatactggc tgtacagtaa ggaactacca cagccaggag cttctaactt 102480 ccaaatttgg cagcagaagg cagctttggc cttgcctaac tgggtgggcc cctctgccaa 102540 gaaccttcac ccactgcttt ttgactatac tagacaaaag gaaggaagaa tggaggacga 102600 ttaacactgc aaagcagtgc atctgaagat aaacgggaag gctgcatctt tctgtttgaa 102660 gattaattat ttttattatt atttctttaa gagacagggt ctcactctgt tgcccaggct 102720 acagtgcagt ggtgcagtca tagctcactg cagcctcaaa ctcctgggct caaatgatct 102780 ccctgccttg gcctcccaaa gtgctgggat cacagccgtg agccaccaca ccctgcaaga 102840 tcaattcttt aacaaattcc aattttatgc aacgtctact cagaggaaaa aaaaaaaaag 102900 tcaccaaagt gttatttttc aatgtgtgcc aggcggtaac agctcctgtt ccaagtctcc 102960 ggccgcatac ctgggaagct gggggatggc cagcgcctcc agggtggagg tcacctctgc 103020 tatgcacagc agcgcgcttc ccaagacatt cttctcctcc tttctctctg gagcaatcag 103080 tttcacagca gtgctcagca ctgggacaaa aggatctgga ttttctgcac caaaattctt 103140 gcataaaagc tttaaggtat acaacgctgt ctgtctgttg attgcttgtt cttcttcccc 103200 ttcctttttc ttacgctgca caatggccaa aaggtctgga accagtttta ggaaacgggt 103260 aacctgaagg ggacagccag aatccccaaa tcattaaagc tgcaaaaaat gtttgtccat 103320 tttcccattg tcacagcttg agattgtcta aatggaaatc agactcgggg gtcctgagtc 103380 acacagtcat gctaagcgat gtgcatgttc tagccagtgt ttcacttata caaagcaccc 103440 actgatctgg agtaaaaggg acttagaact atgctaaggc taaggccacg taagctctgt 103500 agtaagcaag aattccacta ggctgaaatt ccattctaag agctcttaca acacacatat 103560 attcccgtta gaattaacgt cacattttaa aacatgtcat ggtattatat tcagataata 103620 atatacttca atttgaaatt gtaccactag agaaattgaa gggagttaaa tgcagctctt 103680 tgataaagca aagtacagta aatgggtgtg tcctgggtct tcactcacta ttgtcttctt 103740 ccaggatata ttttgctgca gcttgttatt caaaaggtcc agcgctttgc ggcgaacaga 103800 tggcagggga ttgcccacca gccctctgat cacaggaatg aatgtctctg tgggcagcaa 103860 ggcattgacc taaagagaaa ttttatattt aacatgaaaa gaaaaacaaa ttaaaaaaaa 103920 aatcaacttc aattaagaca gactgctgtc cactgcacac ctccaggcac caggcacttc 103980 cacacacatt ttcttattta attcttaaaa taacctttca ggtaggcatt accaaccaca 104040 cattatcgaa caaaacaaaa gcctgatgtc aggaggaagt gccaaaggca tgcagctaaa 104100 tgactgagct agatttgaat cagcaatcct aacttcgagg ccagtgatat gtatgtaata 104160 tacttcatac ttttatttta ttccacttga ataaagtaga acagtatata ttatatgact 104220 taattattaa aatatacgag gtacatgttc tcataactgg taaggaaaca attttttcca 104280 gacaaatcta tttctagtca tcaagagatt gttttctaag aaaaatctga gcttcattat 104340 attcataaaa ggaattgcta agtttattct taaaaacttt acataatttc acaataattt 104400 aaaaaacagc aacaaaacag taattccagg gagaaatgaa cacctacctt atctaacagg 104460 tcgtaagctt tactaaggag cgcgcgccag aacttcacgg tgagtttgtc tgcgttcctt 104520 tccatggact gtgcaactgc actgatatag ccgagaacgg tctccagcaa cctgaaacac 104580 agaggctcgc tcagcaaacg gcagctgaag aaactcagag aacttgttca tgtctacctt 104640 atgctaaatg tttcaagtag aaagacgagt taaataattc tgtactaaat tatttcaaaa 104700 actactcgga aagaaaggaa atgagggatt attgccatag acagagatca tcaagaagta 104760 actaggcgct tctgtgcaga agcatcgacc tcgctcagac tctgtgaggt gctgaataag 104820 caacagatgc tgaaagcgtt taaggaactc actcatatct agctcatgct cagtggatct 104880 cactgggctg tccaagtggg gtgttcaggg agttatggcc ctaggttaat ggcaggtgtg 104940 tgcgtgcaca cacacacagg cacacacacg cacacataca catgcacaca caccatacac 105000 catttatata aagagaaata ttaatagaaa tgaacatata acccacttct ttcacattat 105060 taggagacaa aaaaaaagac tacaaacttc aaataacttg taattagaaa agcacacacc 105120 aaattccaac acagctgcca ctggagatcc ccccactgct gccagcctga ggggggagct 105180 agagggaaga gtggagacag aagttgacac cgcacagcag aggaggggag aagggggcgc 105240 agacaaaatc agctccaaaa acgaaagtcc tacgcatagc gctacaagtc agcccacagg 105300 actggaactc agcagctcac attcctggct gcagggcagg cactttccag tggaaggggc 105360 aggacagtgg ccctgggaat gccatgcatc tgaaaaggag gtacacagca aggccaggag 105420 gcaaaccccg aggacatggg agagaaagga aaattcctgc acccaaatat ataatggcag 105480 catatggatt agaatccacg gaataaagaa ttcatgagcc catagaaatc agggccagat 105540 tgagacacta aacagatact gcaactcaat acaatacaca gacttgacat ggatcatgat 105600 gcagaaacac atgcggtgta aaggacagtg ttgggataat tagggagact ggagtatgaa 105660 ctgtagatta catcactgga ttggatcaat gttaaatttt ctgaatttga tcaatgtact 105720 gtggttttat aagaacatct cttattctta gagacataat gtatatgatt tactttcaaa 105780 tggctcagag aaaaaaccct acatagggag aacgctaagg caaatgtggc agaaagtatt 105840 atcaaatggt gaacctggtt gtaaagagta tatgaatttt ctgtactgtt tttccaggtt 105900 ttctataagt ttgaagtcat ttccaaataa aaagtaaaaa aagaaaagga aacatacctc 105960 tcttcaaggc cttttaaaat ctcaggacca ccactctcaa ctacctaatt tttaaagaag 106020 acgtcattag aacggtatgg aagtcaataa taaaagtcat ttcaagtcag ttcaatgaaa 106080 ctcggaccat tcactgaaac cttccacagc aactgttttc tgacattaca atttaatcag 106140 gttcatagca tcttcattat actgtagtaa ctctatttct cttaatttat tttaattata 106200 ttctactggt agtatctaaa aagtactaca atggttcaga aaaatacagc aatcaacact 106260 caattagcac taccgaattc tatgacatgc tgatctggtg agctcacata tcctttgttg 106320 agaagttaaa cattacagat tcagctggaa tcccccaagt actgctcctt ggtcctattc 106380 tccctctacc ccaagcccca caaacaaaac catcatccca aatctgcttc caaatgtttc 106440 aaacactaca tatcacggaa caacatgttt ttctggaaac atatttttga gatctatgca 106500 tggtgactta tgttctagtt ccttcatttt aactgcatat gatattcctc tataaatacc 106560 acttatctat ccatttgcct ctgttgttag atgtttagtt tatgtccatt ttttcccctt 106620 ttactaataa tgctagagaa gaacattttt atgtcccttt gatcatcttg ggaagttttt 106680 acagcatata tacctaagga agggaatgac cagatcacag gaattactgg aactttcaac 106740 ctcatg 106746 13 132 PRT Homo sapiens MISC_FEATURE (1)..(132) amino acid sequence of leg2 13 Met Thr Gly Glu Leu Glu Val Lys Asn Met Asp Met Lys Pro Gly Ser 1 5 10 15 Thr Leu Lys Ile Thr Gly Ser Ile Ala Asp Gly Thr Asp Gly Phe Val 20 25 30 Ile Asn Leu Gly Gln Gly Thr Asp Lys Leu Asn Leu His Phe Asn Pro 35 40 45 Arg Phe Ser Glu Ser Thr Ile Val Cys Asn Ser Leu Asp Gly Ser Asn 50 55 60 Trp Gly Gln Glu Gln Arg Glu Asp His Leu Cys Phe Ser Pro Gly Ser 65 70 75 80 Glu Val Lys Phe Thr Val Thr Phe Glu Ser Asp Lys Phe Lys Val Lys 85 90 95 Leu Pro Asp Gly His Glu Leu Thr Phe Pro Asn Arg Leu Gly His Ser 100 105 110 His Leu Ser Tyr Leu Ser Val Arg Gly Gly Phe Asn Met Ser Ser Phe 115 120 125 Lys Leu Lys Glu 130 14 134 PRT Homo sapiens MISC_FEATURE (1)..(134) amino acid sequence of leg1 14 Ala Cys Gly Leu Val Ala Ser Asn Leu Asn Leu Lys Pro Gly Glu Cys 1 5 10 15 Leu Arg Val Arg Gly Glu Val Ala Pro Asp Ala Lys Ser Phe Val Leu 20 25 30 Asn Leu Gly Lys Asp Ser Asn Asn Leu Cys Leu His Phe Asn Pro Arg 35 40 45 Phe Asn Ala His Gly Asp Ala Asn Thr Ile Val Cys Asn Ser Lys Asp 50 55 60 Gly Gly Ala Trp Gly Thr Glu Gln Arg Glu Ala Val Phe Pro Phe Gln 65 70 75 80 Pro Gly Ser Val Ala Glu Val Cys Ile Thr Phe Asp Gln Ala Asn Leu 85 90 95 Thr Val Lys Leu Pro Asp Gly Tyr Glu Phe Lys Phe Pro Asn Arg Leu 100 105 110 Asn Leu Glu Ala Ile Asn Tyr Met Ala Ala Asp Gly Asp Phe Lys Ile 115 120 125 Lys Cys Val Ala Phe Asp 130 15 316 PRT Homo sapiens MISC_FEATURE (1)..(316) amino acid sequence of PCTA 15 Met Leu Ser Leu Asn Asn Leu Gln Asn Ile Ile Tyr Asn Pro Val Ile 1 5 10 15 Pro Tyr Val Gly Thr Ile Pro Asp Gln Leu Asp Pro Gly Thr Leu Ile 20 25 30 Val Ile Cys Gly His Val Pro Ser Asp Ala Asp Arg Phe Gln Val Asp 35 40 45 Leu Gln Asn Gly Ser Ser Val Lys Pro Arg Ala Asp Val Ala Phe His 50 55 60 Phe Asn Pro Arg Phe Lys Arg Ala Gly Cys Ile Val Cys Asn Thr Leu 65 70 75 80 Ile Asn Glu Lys Trp Gly Arg Glu Glu Ile Thr Tyr Asp Thr Pro Phe 85 90 95 Lys Arg Glu Lys Ser Phe Glu Ile Val Ile Met Val Leu Lys Asp Lys 100 105 110 Phe Gln Val Ala Val Asn Gly Lys His Thr Leu Leu Tyr Gly His Arg 115 120 125 Ile Gly Pro Glu Lys Ile Asp Thr Leu Gly Ile Tyr Gly Lys Val Asn 130 135 140 Ile His Ser Ile Gly Phe Ser Phe Ser Ser Asp Leu Gln Ser Thr Gln 145 150 155 160 Ala Ser Ser Leu Glu Leu Thr Glu Ile Ser Arg Glu Asn Val Pro Lys 165 170 175 Ser Gly Thr Pro Gln Leu Ser Leu Pro Phe Ala Ala Arg Leu Asn Thr 180 185 190 Pro Met Gly Pro Gly Arg Thr Val Val Val Lys Gly Glu Val Asn Ala 195 200 205 Asn Ala Lys Ser Phe Asn Val Asp Leu Leu Ala Gly Lys Ser Lys Asp 210 215 220 Ile Ala Leu His Leu Asn Pro Arg Leu Asn Ile Lys Ala Phe Val Arg 225 230 235 240 Asn Ser Phe Leu Gln Glu Ser Trp Gly Glu Glu Glu Arg Asn Ile Thr 245 250 255 Ser Phe Pro Phe Ser Pro Gly Met Tyr Phe Glu Met Ile Ile Tyr Cys 260 265 270 Asp Val Arg Glu Phe Lys Val Ala Val Asn Gly Val His Ser Leu Glu 275 280 285 Tyr Lys His Arg Phe Lys Glu Leu Ser Ser Ile Asp Thr Leu Glu Ile 290 295 300 Asn Gly Asp Ile His Leu Leu Glu Val Arg Ser Trp 305 310 315 16 358 PRT Homo sapiens MISC_FEATURE (1)..(358) amino acid sequence of PCTA.var 16 Met Leu Ser Leu Asn Asn Leu Gln Asn Ile Ile Tyr Asn Pro Val Ile 1 5 10 15 Pro Tyr Val Gly Thr Ile Pro Asp Gln Leu Asp Pro Gly Thr Leu Ile 20 25 30 Val Ile Cys Gly His Val Pro Ser Asp Ala Asp Arg Phe Gln Val Asp 35 40 45 Leu Gln Asn Gly Ser Ser Val Lys Pro Arg Ala Asp Val Ala Phe His 50 55 60 Phe Asn Pro Arg Phe Lys Arg Ala Gly Cys Ile Val Cys Asn Thr Leu 65 70 75 80 Ile Asn Glu Lys Trp Gly Arg Glu Glu Ile Thr Tyr Asp Thr Pro Phe 85 90 95 Lys Arg Glu Lys Ser Phe Glu Ile Val Ile Met Val Leu Lys Asp Lys 100 105 110 Phe Gln Val Ala Val Asn Gly Lys His Thr Leu Leu Tyr Gly His Arg 115 120 125 Ile Gly Pro Glu Lys Ile Asp Thr Leu Gly Ile Tyr Gly Lys Val Asn 130 135 140 Ile His Ser Ile Gly Phe Ser Phe Ser Ser Asp Leu Gln Ser Thr Gln 145 150 155 160 Ala Ser Ser Leu Glu Leu Thr Glu Ile Ser Arg Glu Asn Val Pro Lys 165 170 175 Ser Gly Thr Pro Gln Leu Pro Ser Asn Arg Gly Gly Asp Ile Ser Lys 180 185 190 Ile Ala Pro Arg Thr Val Tyr Thr Lys Ser Lys Asp Ser Thr Val Asn 195 200 205 His Thr Leu Thr Cys Thr Lys Ile Pro Pro Met Asn Tyr Val Ser Lys 210 215 220 Ser Leu Pro Phe Ala Ala Arg Leu Asn Thr Pro Met Gly Pro Gly Arg 225 230 235 240 Thr Val Val Val Lys Gly Glu Val Asn Ala Asn Ala Lys Ser Phe Asn 245 250 255 Val Asp Leu Leu Ala Gly Lys Ser Lys Asp Ile Ala Leu His Leu Asn 260 265 270 Pro Arg Leu Asn Ile Lys Ala Phe Val Arg Asn Ser Phe Leu Gln Glu 275 280 285 Ser Trp Gly Glu Glu Glu Arg Asn Ile Thr Ser Phe Pro Phe Ser Pro 290 295 300 Gly Met Tyr Phe Glu Met Ile Ile Tyr Cys Asp Val Arg Glu Phe Lys 305 310 315 320 Val Ala Val Asn Gly Val His Ser Leu Glu Tyr Lys His Arg Phe Lys 325 330 335 Glu Leu Ser Ser Ile Asp Thr Leu Glu Ile Asn Gly Asp Ile His Leu 340 345 350 Leu Glu Val Arg Ser Trp 355 17 315 PRT Mus musculus MISC_FEATURE (1)..(315) amino acid sequence of PCTA.mus 17 Met Leu Ser Leu Asn Asn Leu Gln Asn Ile Ile Tyr Asn Pro Ile Ile 1 5 10 15 Pro Tyr Val Gly Thr Ile Thr Glu Gln Leu Lys Pro Gly Ser Leu Ile 20 25 30 Val Ile Arg Gly His Val Pro Lys Asp Ser Glu Arg Phe Gln Val Asp 35 40 45 Phe Gln Leu Gly Asn Ser Leu Lys Pro Arg Ala Asp Val Ala Phe His 50 55 60 Phe Asn Pro Arg Phe Lys Arg Ser Ser Cys Ile Val Cys Asn Thr Leu 65 70 75 80 Thr Gln Glu Lys Trp Gly Trp Glu Glu Ile Thr Tyr Asp Met Pro Phe 85 90 95 Arg Lys Glu Lys Ser Phe Glu Ile Val Phe Met Val Leu Lys Asn Lys 100 105 110 Phe Gln Val Ala Val Asn Gly Arg His Val Leu Leu Tyr Ala His Arg 115 120 125 Ile Ser Pro Glu Gln Ile Asp Thr Val Gly Ile Tyr Gly Lys Val Asn 130 135 140 Ile His Ser Ile Gly Phe Arg Phe Ser Ser Asp Leu Gln Ser Met Glu 145 150 155 160 Thr Ser Ala Leu Gly Leu Thr Gln Ile Asn Arg Glu Asn Ile Gln Lys 165 170 175 Pro Gly Lys Leu Gln Leu Ser Leu Pro Phe Glu Ala Arg Leu Asn Ala 180 185 190 Ser Met Gly Pro Gly Arg Thr Val Val Ile Lys Gly Glu Val Asn Thr 195 200 205 Asn Ala Arg Ser Phe Asn Val Asp Leu Val Ala Gly Lys Thr Arg Asp 210 215 220 Ile Ala Leu His Leu Asn Pro Arg Leu Asn Lys Ala Phe Val Arg Asn 225 230 235 240 Ser Phe Leu Gln Asp Ala Trp Gly Glu Glu Glu Arg Asn Ile Thr Cys 245 250 255 Phe Pro Phe Ser Ser Gly Met Tyr Phe Glu Met Ile Ile Tyr Cys Asp 260 265 270 Val Arg Glu Phe Lys Val Ala Ile Asn Gly Val His Ser Leu Glu Tyr 275 280 285 Lys His Arg Phe Lys Asp Leu Ser Ser Ile Asp Thr Leu Ser Val Asp 290 295 300 Gly Asp Ile Arg Leu Leu Asp Val Arg Ser Trp 305 310 315 18 355 PRT Homo sapiens MISC_FEATURE (1)..(355) amino acid sequence of gal9-1 18 Met Ala Phe Ser Gly Ser Gln Ala Pro Tyr Leu Ser Pro Ala Val Pro 1 5 10 15 Phe Ser Gly Thr Ile Gln Gly Gly Leu Gln Asp Gly Leu Gln Ile Thr 20 25 30 Val Asn Gly Thr Val Leu Ser Ser Ser Gly Thr Arg Phe Ala Val Asn 35 40 45 Phe Gln Thr Gly Phe Ser Gly Asn Asp Ile Ala Phe His Phe Asn Pro 50 55 60 Arg Phe Glu Asp Gly Gly Tyr Val Val Cys Asn Thr Arg Gln Asn Gly 65 70 75 80 Ser Trp Gly Pro Glu Glu Arg Lys Thr His Met Pro Phe Gln Lys Gly 85 90 95 Met Pro Phe Asp Leu Cys Phe Leu Val Gln Ser Ser Asp Phe Lys Val 100 105 110 Met Val Asn Gly Ile Leu Phe Val Gln Tyr Phe His Arg Val Pro Phe 115 120 125 His Arg Val Asp Thr Ile Ser Val Asn Gly Ser Val Gln Leu Ser Tyr 130 135 140 Ile Ser Phe Gln Asn Pro Arg Thr Val Pro Val Gln Pro Ala Phe Ser 145 150 155 160 Thr Val Pro Phe Ser Gln Pro Val Cys Phe Pro Pro Arg Pro Arg Gly 165 170 175 Arg Arg Gln Lys Pro Pro Gly Val Trp Pro Ala Asn Pro Ala Pro Ile 180 185 190 Thr Gln Thr Val Ile His Thr Val Gln Ser Ala Pro Gly Gln Met Phe 195 200 205 Ser Thr Pro Ala Ile Pro Pro Met Met Tyr Pro His Pro Ala Tyr Pro 210 215 220 Met Pro Phe Ile Thr Thr Ile Leu Gly Gly Leu Tyr Pro Ser Lys Ser 225 230 235 240 Ile Leu Leu Ser Gly Thr Val Leu Pro Ser Ala Gln Arg Phe His Ile 245 250 255 Asn Leu Cys Ser Gly Asn His Ile Ala Phe His Leu Asn Pro Arg Phe 260 265 270 Asp Glu Asn Ala Val Val Arg Asn Thr Gln Ile Asp Asn Ser Trp Gly 275 280 285 Ser Glu Glu Arg Ser Leu Pro Arg Lys Met Pro Phe Val Arg Gly Gln 290 295 300 Ser Phe Ser Val Trp Ile Leu Cys Glu Ala His Cys Leu Lys Val Ala 305 310 315 320 Val Asp Gly Gln His Leu Phe Glu Tyr Tyr His Arg Leu Arg Asn Leu 325 330 335 Pro Thr Ile Asn Arg Leu Glu Val Gly Gly Asp Ile Gln Leu Thr His 340 345 350 Val Gln Thr 355 19 323 PRT Homo sapiens MISC_FEATURE (1)..(323) amino acid sequence of gal 19 Met Ala Phe Ser Gly Ser Gln Ala Pro Tyr Leu Ser Pro Ala Val Pro 1 5 10 15 Phe Ser Gly Thr Ile Gln Gly Gly Leu Gln Asp Gly Leu Gln Ile Thr 20 25 30 Val Asn Gly Thr Val Leu Ser Ser Ser Gly Thr Arg Phe Ala Val Asn 35 40 45 Phe Gln Thr Gly Phe Ser Gly Asn Asp Ile Ala Phe His Phe Asn Pro 50 55 60 Arg Phe Glu Asp Gly Gly Tyr Val Val Cys Asn Thr Arg Gln Asn Gly 65 70 75 80 Ser Trp Gly Pro Glu Glu Arg Arg Thr His Met Pro Phe Gln Lys Gly 85 90 95 Met Pro Phe Asp Leu Cys Phe Leu Val Gln Ser Ser Asp Phe Lys Val 100 105 110 Met Val Asn Gly Ile Leu Phe Val Gln Tyr Phe His Arg Val Pro Phe 115 120 125 His Arg Val Asp Thr Ile Phe Val Asn Gly Ser Val Gln Leu Ser Tyr 130 135 140 Ile Ser Phe Gln Pro Pro Gly Val Trp Pro Ala Asn Pro Ala Pro Ile 145 150 155 160 Thr Gln Thr Val Ile His Thr Val Gln Ser Ala Pro Gly Gln Met Phe 165 170 175 Ser Thr Pro Ala Ile Pro Pro Met Met Tyr Pro His Pro Ala Tyr Pro 180 185 190 Met Pro Phe Ile Thr Thr Ile Leu Gly Gly Leu Tyr Pro Ser Lys Ser 195 200 205 Ile Leu Leu Ser Gly Thr Val Leu Pro Ser Ala Gln Arg Phe His Ile 210 215 220 Asn Leu Cys Ser Gly Asn His Ile Ala Phe His Leu Asn Leu Arg Phe 225 230 235 240 Asp Glu Asn Ala Val Val Arg Asn Thr Gln Ile Asp Asn Ser Trp Gly 245 250 255 Ser Glu Glu Arg Ser Leu Pro Arg Lys Met Pro Phe Val Arg Gly Gln 260 265 270 Ser Phe Ser Val Trp Ile Leu Cys Gly Ala His Cys Leu Lys Val Ala 275 280 285 Val Asp Gly Gln His Leu Phe Glu Tyr Tyr His Arg Leu Arg Asn Leu 290 295 300 Pro Thr Ile Asn Arg Leu Glu Val Gly Gly Asp Ile Gln Leu Thr His 305 310 315 320 Val Gln Thr 20 135 PRT Homo sapiens MISC_FEATURE (1)..(135) amino acid sequence of leg7 20 Ser Asn Val Pro His Lys Ser Ser Leu Pro Glu Gly Ile Arg Pro Gly 1 5 10 15 Thr Val Leu Arg Ile Arg Gly Leu Val Pro Pro Asn Ala Ser Arg Phe 20 25 30 His Val Asn Leu Leu Cys Gly Glu Glu Gln Gly Ser Asp Ala Ala Leu 35 40 45 His Phe Asn Pro Arg Leu Asp Thr Ser Glu Val Val Phe Asn Ser Lys 50 55 60 Glu Gln Gly Ser Trp Gly Arg Glu Glu Arg Gly Pro Gly Val Pro Phe 65 70 75 80 Gln Arg Gly Gln Pro Phe Glu Val Leu Ile Ile Ala Ser Asp Asp Gly 85 90 95 Phe Lys Ala Val Val Gly Asp Ala Gln Tyr His His Phe Arg His Arg 100 105 110 Leu Pro Leu Ala Arg Val Arg Leu Val Glu Val Gly Gly Asp Val Gln 115 120 125 Leu Asp Ser Val Arg Ile Phe 130 135 21 323 PRT Homo sapiens MISC_FEATURE (1)..(323) amino acid sequence of gal4 21 Met Ala Tyr Val Pro Ala Pro Gly Tyr Gln Pro Thr Tyr Asn Pro Thr 1 5 10 15 Leu Pro Tyr Tyr Gln Pro Ile Pro Gly Gly Leu Asn Val Gly Met Ser 20 25 30 Val Tyr Ile Gln Gly Val Ala Ser Glu His Met Lys Arg Phe Phe Val 35 40 45 Asn Phe Val Val Gly Gln Asp Pro Gly Ser Asp Val Ala Phe His Phe 50 55 60 Asn Pro Arg Phe Asp Gly Trp Asp Lys Val Val Phe Asn Thr Leu Gln 65 70 75 80 Gly Gly Lys Trp Gly Ser Glu Glu Arg Lys Arg Ser Met Pro Phe Lys 85 90 95 Lys Gly Ala Ala Phe Glu Leu Val Phe Ile Val Leu Ala Glu His Tyr 100 105 110 Lys Val Val Val Asn Gly Asn Pro Phe Tyr Glu Tyr Gly His Arg Leu 115 120 125 Pro Leu Gln Met Val Thr His Leu Gln Val Asp Gly Asp Leu Gln Leu 130 135 140 Gln Ser Ile Asn Phe Ile Gly Gly Gln Pro Leu Arg Pro Gln Gly Pro 145 150 155 160 Pro Met Met Pro Pro Tyr Pro Gly Pro Gly His Cys His Gln Gln Leu 165 170 175 Asn Ser Leu Pro Thr Met Glu Gly Pro Pro Thr Phe Asn Pro Pro Val 180 185 190 Pro Tyr Phe Gly Arg Leu Gln Gly Gly Leu Thr Ala Arg Arg Thr Ile 195 200 205 Ile Ile Lys Gly Tyr Val Pro Pro Thr Gly Lys Ser Phe Ala Ile Asn 210 215 220 Phe Lys Val Gly Ser Ser Gly Asp Ile Ala Leu His Ile Asn Pro Arg 225 230 235 240 Met Gly Asn Gly Thr Val Val Arg Asn Ser Leu Leu Asn Gly Ser Trp 245 250 255 Gly Ser Glu Glu Lys Lys Ile Thr His Asn Pro Phe Gly Pro Gly Gln 260 265 270 Phe Phe Asp Leu Ser Ile Arg Cys Gly Leu Asp Arg Phe Lys Val Tyr 275 280 285 Ala Asn Gly Gln His Leu Phe Asp Phe Ala His Arg Leu Ser Ala Phe 290 295 300 Gln Arg Val Asp Thr Leu Glu Ile Gln Gly Asp Val Thr Leu Ser Tyr 305 310 315 320 Val Gln Ile 22 466 PRT Artificial Sequence conserved sequence among SEQ ID NOs. 13-21 22 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Pro Xaa Xaa Pro Xaa Xaa Xaa Xaa Ile Pro Xaa Gly Leu Xaa Pro 115 120 125 Gly Xaa Xaa Xaa Xaa Ile Xaa Gly Xaa Val Xaa Pro Xaa Xaa Ala Xaa 130 135 140 Arg Phe Xaa Val Asn Leu Xaa Xaa Gly Xaa Xaa Xaa Xaa Xaa Gly Xaa 145 150 155 160 Asp Xaa Ala Phe His Phe Asn Pro Arg Phe Xaa Xaa Xaa Xaa Xaa Xaa 165 170 175 Xaa Xaa Val Val Cys Asn Thr Xaa Xaa Xaa Gly Xaa Trp Gly Xaa Glu 180 185 190 Glu Arg Xaa Xaa Xaa Xaa Pro Phe Xaa Xaa Gly Xaa Xaa Phe Glu Xaa 195 200 205 Xaa Xaa Xaa Val Xaa Xaa Asp Xaa Phe Lys Val Xaa Val Asn Gly Xaa 210 215 220 Xaa Xaa Xaa Xaa Tyr Xaa His Arg Leu Xaa Pro Leu Xaa Xaa Val Xaa 225 230 235 240 Xaa Xaa Xaa Val Xaa Gly Asp Val Gln Leu Xaa Ser Ile Xaa Phe Xaa 245 250 255 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 260 265 270 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 275 280 285 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 290 295 300 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 305 310 315 320 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa 325 330 335 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 340 345 350 Gly Xaa Val Xaa Xaa Xaa Xaa Xaa Xaa Phe Xaa Xaa Xaa Xaa Xaa Xaa 355 360 365 Xaa Xaa Xaa Xaa Xaa Ile Ala Xaa His Xaa Asn Xaa Arg Xaa Xaa Xaa 370 375 380 Xaa Xaa Xaa Val Arg Asn Xaa Xaa Xaa Xaa Xaa Xaa Trp Gly Xaa Glu 385 390 395 400 Glu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro Phe Xaa Xaa Gly Xaa Xaa Phe 405 410 415 Xaa Xaa Xaa Ile Xaa Cys Xaa Xaa Xaa Xaa Xaa Lys Val Xaa Xaa Xaa 420 425 430 Gly Xaa His Xaa Xaa Xaa Xaa Xaa His Arg Xaa Xaa Xaa Xaa Xaa Xaa 435 440 445 Xaa Xaa Xaa Leu Xaa Xaa Xaa Gly Asp Xaa Xaa Leu Xaa Xaa Val Xaa 450 455 460 Xaa Xaa 465 

What is claimed is:
 1. A method of genotyping comprising determining the identity of a nucleotide at a biallelic marker of SEQ ID NO: 12, or the complement of said nucleotide, in a biological sample, wherein said biallelic marker is selected from the group consisting of biallelic markers of SEQ ID No:1 located at positions 402 (A2), 67092 (A30), 68525 (A41), 82234 (A55), 82393 (A57), and 87713 (A75).
 2. The method according to claim 1, wherein said biological sample is derived from a single subject.
 3. The method according to claim 2, wherein the identity of the nucleotides at said biallelic marker is determined for both copies of said biallelic marker present in said individual's genome.
 4. A method of estimating the frequency of a haplotype for a set of biallelic markers of SEQ ID NO: 12 in a population, comprising: a) genotyping at least one biallelic marker of SEQ ID NO: 12 according to claim 2 for each individual in said population; b) genotyping a second biallelic marker of SEQ ID NO: 12 by determining the identity of the nucleotide at said second biallelic marker for both copies of said second biallelic marker present in the genome of each individual in said population; and c) applying a haplotype determination method to the identities of the nucleotides determined in steps a) and b) to obtain an estimate of said frequency.
 5. The method according to claim 4, wherein said haplotype determination method is selected from the group consisting of asymmetric PCR amplification, double PCR amplification of specific alleles, the Clark algorithm, or an expectation-maximization algorithm.
 6. A method of detecting an association between a haplotype and a trait, comprising the steps of: a) estimating the frequency of at least one haplotype in a trait positive population according to the method of claim 4; b) estimating the frequency of said haplotype in a control population according to the method of claim 4; and c) determining whether a statistically significant association exists between said haplotype and said trait, wherein said trait is familial or sporadic prostate cancer.
 7. The method according to claim 6, wherein said control population is a trait negative population.
 8. The method according to claim 6, wherein said control population is a random population.
 9. The method according to claim 1, wherein said biological sample is derived from multiple subjects.
 10. The method according to claim 1, further comprising amplifying a portion of said sequence comprising the biallelic marker prior to said determining step.
 11. The method according to claim 10, wherein said amplifying is performed by PCR.
 12. The method according to claim 1, wherein said determining is performed by a hybridization assay.
 13. The method according to claim 1, wherein said determining is performed by a sequencing assay.
 14. The method according to claim 1, wherein said determining is performed by a microsequencing assay.
 15. The method according to claim 1, wherein said determining is performed by an enzyme-based mismatch detection assay.
 16. A method of estimating the frequency of an allele of a biallelic marker of SEQ ID NO: 12 in a population comprising: a) genotyping individuals from said population for said biallelic marker according to the method of claim 1; and b) determining the proportional representation of said biallelic marker in said population.
 17. A method of detecting an association between a genotype and a trait, comprising the steps of: a) performing the method of claim 16 to determine the frequency of at least one biallelic marker of SEQ ID NO: 12 in trait positive population; b) performing the method of claim 16 to determine the frequency of at least one biallelic marker of SEQ ID NO: 12 in a control population; and c) determining whether a statistically significant association exists between said genotype and said trait, wherein said trait is familial or sporadic prostate cancer.
 18. The method according to claim 17, wherein said genotyping steps a) and b) are performed on a single pooled biological sample derived from each of said populations.
 19. The method according to claim 17, wherein said genotyping steps a) and b) performed separately on biological samples derived from each individual in said populations.
 20. The method according to claim 17, wherein said control population is a trait negative population.
 21. The method according to claim 17, wherein said control population is a random population.
 22. A method of determining whether an individual is at risk of developing familial or sporadic prostate cancer, comprising: a) genotyping at least one biallelic marker of SEQ ID NO: 12 according to the method of claim 3; b) determining if the individual has a biallelic marker or combination of biallelic markers that is associated with familial or sporadic prostate cancer; and c) correlating the result of step b) with a biallelic marker or one or more combinations of biallelic markers that are associated with a risk of developing familial or sporadic prostate cancer; wherein: for familial cases of prostate cancer said biallelic marker or combinations of biallelic markers are selected from the group consisting of: 1) A30 and A41; 2) A30 and A57; 3); A30 and A55; 4) A2 and A41; 5) A2 and A30; 6) A30 and A75; 7) A2 and A55; 8) A2, A30, and A41; 9) A2, A30, and A57; 10) A2, A30, and A55; 11) A30, A41, and A55; 12) A30, A57, and A75; 13) A30, A55, and A75; 14) A30, A41, and A75; 15) A30, A55, and A57; 16) A30, A41, and A57; 17) A2, A30, A55, and A57; 18) A2, A30, A57, and A75; 19) A2, A30, A55, and A75; 20) A2, A30, A41, and A57; 21) A2, A30, A41, and A55; 22) A2, A30, A41, and A75; 23) A30, A55, A57, and A75; 24) A30, A41, A55, and A75; 25) A30, A41, A55, and A57; 26) A30, A41, A57, and A75 and A30; and 27) A30; and for sporadic cases of prostate cancer said combinations of biallelic markers are selected from the group consisting of: 1) A2 and A55; 2) A2 and A57; 3) A41 and A55; 4) A41 and A57; 5) A2 and A41; 6) A30 and A75; 7) A2, A41, and A55; 8) A2, A55, and A57; 9) A2, A41, and A57; 10) A41, A55, and A57; 11) A2, A55, and A75; 12) A30, A41, and A57; 13) A30, A41, and A55; 14) A2, A30, A41, and A57; 15) A2, A30, A41, and A55; 16) A2, A41, A55, and A57; 17) A2, A41, A55, and A75; and 18) A30, A41, A55, and A57. 